Immunogenic trimers

ABSTRACT

The invention relates to PGT121-germline-targeting designs, trimer stabilization designs, combinations of those two, trimers designed with modified surfaces helpful for immunization regimens, other trimer modifications and on development of trimer nanoparticles and methods of making and using the same.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a continuation-in-part of international applicationSerial No. PCT/US2017/023854 filed Mar. 23, 2017, which published as PCTPublication No. WO 2017/165674 on Sep. 28, 2017, which claims benefit ofand priority to U.S. provisional patent application Ser. No. 62/312,190filed Mar. 23, 2016 and Ser. No. 62/384,762 filed Sep. 8, 2016.

All documents cited or referenced herein (“herein cited documents”), andall documents cited or referenced in herein cited documents, togetherwith any manufacturer's instructions, descriptions, productspecifications, and product sheets for any products mentioned herein orin any document incorporated by reference herein, are herebyincorporated herein by reference, and may be employed in the practice ofthe invention. More specifically, all referenced documents areincorporated by reference to the same extent as if each individualdocument was specifically and individually indicated to be incorporatedby reference.

FEDERAL FUNDING LEGEND

This invention was made with government support under Grant Nos.CHAVI-ID 1UM1AI100663 and R01 AI084817 awarded by the National Instituteof Allergy and Infectious Disease. This invention was also made withgovernment support under Grant No. P41GM103393 awarded by the NationalInstitutes of Health, National Institute of General Medical Sciences.The government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 5, 2017, isnamed 43094_99_2041_SL.txt and is 680,679 bytes in size

FIELD OF THE INVENTION

The invention relates to PGT121-germline-targeting designs, trimerstabilization designs, combinations of those two, trimers designed withmodified surfaces helpful for immunization regimens, other trimermodifications and on development of trimer nanoparticles andmembrane-bound trimers.

BACKGROUND OF THE INVENTION

AIDS, or Acquired Immunodeficiency Syndrome, is caused by humanimmunodeficiency virus (HIV) and is characterized by several clinicalfeatures including wasting syndromes, central nervous systemdegeneration and profound immunosuppression that results inopportunistic infections and malignancies. HIV is a member of thelentivirus family of animal retroviruses, which include the visna virusof sheep and the bovine, feline, and simian immunodeficiency viruses(SIV). Two closely related types of HIV, designated HIV-1 and HIV-2,have been identified thus far, of which HIV-1 is by far the most commoncause of AIDS. However, HIV-2, which differs in genomic structure andantigenicity, causes a similar clinical syndrome.

An infectious HIV particle consists of two identical strands of RNA,each approximately 9.2 kb long, packaged within a core of viralproteins. This core structure is surrounded by a phospholipid bilayerenvelope derived from the host cell membrane that also includes virallyencoded membrane proteins (Abbas et al., Cellular and MolecularImmunology, 4th edition, W.B. Saunders Company, 2000, p. 454). The HIVgenome has the characteristic 5′-LTR-Gag-Pol-Env-LTR-3′ organization ofthe retrovirus family. Long terminal repeats (LTRs) at each end of theviral genome serve as binding sites for transcriptional regulatoryproteins from the host and regulate viral integration into the hostgenome, viral gene expression, and viral replication.

The HIV genome encodes several structural proteins. The gag gene encodesstructural proteins of the nucleocapsid core and matrix. The pol geneencodes reverse transcriptase (RT), integrase (IN), and viral protease(PR) enzymes required for viral replication. The tat gene encodes aprotein that is required for elongation of viral transcripts. The revgene encodes a protein that promotes the nuclear export of incompletelyspliced or unspliced viral RNAs. The vif gene product enhances theinfectivity of viral particles. The vpr gene product promotes thenuclear import of viral DNA and regulates G2 cell cycle arrest. The vpuand nef genes encode proteins that down regulate host cell CD4expression and enhance release of virus from infected cells. The envgene encodes the viral envelope glycoprotein that is translated as a160-kilodalton (kDa) precursor (gp160) and cleaved by a cellularprotease to yield the external 120-kDa envelope glycoprotein (gp120) andthe transmembrane 41-kDa envelope glycoprotein (gp41), which arerequired for the infection of cells (Abbas et al., Cellular andMolecular Immunology, 4th edition, W.B. Saunders Company, 2000, pp.454-456). gp140 is a modified form of the Env glycoprotein, whichcontains the external 120-kDa envelope glycoprotein portion and theextracellular part of the gp41 portion of Env and has characteristics ofboth gp120 and gp41. The nef gene is conserved among primatelentiviruses and is one of the first viral genes that is transcribedfollowing infection. In vitro, several functions have been described,including down-regulation of CD4 and MHC class I surface expression,altered T-cell signaling and activation, and enhanced viral infectivity.

HIV infection initiates with gp120 on the viral particle binding to theCD4 and chemokine receptor molecules (e.g., CXCR4, CCR5) on the cellmembrane of target cells such as CD4+ T-cells, macrophages and dendriticcells. The bound virus fuses with the target cell and reversetranscribes the RNA genome. The resulting viral DNA integrates into thecellular genome, where it directs the production of new viral RNA, andthereby viral proteins and new virions. These virions bud from theinfected cell membrane and establish productive infections in othercells. This process also kills the originally infected cell. HIV canalso kill cells indirectly because the CD4 receptor on uninfectedT-cells has a strong affinity for gp120 expressed on the surface ofinfected cells. In this case, the uninfected cells bind, via the CD4receptor-gp120 interaction, to infected cells and fuse to form asyncytium, which cannot survive. Destruction of CD4+T-lymphocytes, whichare critical to immune defense, is a major cause of the progressiveimmune dysfunction that is the hallmark of AIDS disease progression. Theloss of CD4+ T cells seriously impairs the body's ability to fight mostinvaders, but it has a particularly severe impact on the defensesagainst viruses, fungi, parasites and certain bacteria, includingmycobacteria.

Viruses have evolved a variety of mechanisms to escape antibodyrecognition, many of which involve features of the viral surfaceproteins, such as high variability, steric occlusion, and glycancoating. For HIV, the dense shield of glycans that decorate the viralEnv protein was once believed to be refractory to antibody recognition,shielding conserved protein epitopes of important functionalsignificance whose greater exposure would result in increasedsusceptibility to antibody neutralization.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

The invention relates to PGT121-germline-targeting designs, trimerstabilization designs, combinations of those two, trimers designed withmodified surfaces helpful for immunization regimens, other types oftrimer modifications (see, for example, IV: Examples of trimers withcombined germline-targeting mutations and stabilization mutations andVI: Additional trimer modifications that add functionality and that canbe combined with other types of modifications as described herein) andon development of trimer nanoparticles and membrane-bound trimers. Theinvention also encompasses combinations of any of the herein describedmodifications, such as but not limited to, combinations of stabilizationand modified surfaces with nanoparticles or membrane-bound trimers.

The HIV envelope protein trimer is the target of broadly neutralizingantibodies (bNAbs). The high mannose patch, including the N332-linkedglycan at the base of the V3 loop of gp120, is frequently targeted bybnAbs during natural infection and hence is an appealing vaccineepitope. Germline targeting has potential to initiate the elicitation ofN332-dependent bnAbs by vaccination, but no immunogen has been reportedto bind germline-reverted precursors of N332-dependent bnAbs.

PGT121 is one of the most broad and potent of the N332-dependent bnAbs.A structural comparison of the mature PGT121 bnAb with itsgermline-reverted precursors suggested structural regions in BG505 SOSIPthat should be modified to generate affinity for the germline. Guided bythis structural analysis, Applicants employed mammalian cell surfacedisplay to engineer BG505 SOSIP trimer variants with appreciableaffinity for germline-reverted PGT121. In the process of developing thegermline-targeting immunogen, Applicants produced a series of lessmutated trimer immunogens with varying affinities for germline PGT121and for partially mutated variants of PGT121. Informed by the bindingaffinities and structural features of the intermediate immunogens,Applicants then postulated sequences of boosting immunogens to guidematuration along different paths following germline activation.

The invention also encompasses a protein having at least 90% homology oridentity with the sequence of the protein of any one of the trimersdisclosed herein. The invention also encompasses a protein having atleast 95% homology or identity with the sequence of the protein of anyone of trimers disclosed herein.

The invention also encompasses any nucleic acid encoding the protein ofany one of the trimers disclosed herein. The invention also encompassesa nucleic acid having at least 90% or 95% homology or identity with thesequence of said nucleic acid.

The present invention also encompasses methods for eliciting an immuneresponse which may comprise systemically administering to an animal inneed thereof an effective amount of the protein of any one of thetrimers disclosed herein. The animal may be a mammal, advantageously ahuman.

Accordingly, it is an object of the invention not to encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. § 112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product. It may be advantageous in thepractice of the invention to be in compliance with Art. 53(c) EPC andRule 28(b) and (c) EPC. Nothing herein is to be construed as a promise.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings.

FIG. 1 depicts a mammalian display/directed evolution overview.

FIG. 2 depicts a mammalian display strategy using partially mutatedPGT121 intermediate antibodies

FIG. 3 depicts binding affinities (SPR Kd (nM)) of germline-targetinggp120s (monomeric gp120 used for SPR, but SOSIP used for immunization)NB, no binding; NB**, no binding at 150 uM.

FIG. 4 depicts biophysical properties of a SOSIP trimer engineered bymammalian display directed evolution and having improvedthermostability, yield and antigenic profile. A BG505 SOSIP trimer wholegene saturation mutagenesis library was FACS sorted on the surface ofmammalian cells for reduced binding to B6 and 4025 and enhanced bindingto PGT145 and PGT151. Additional combinatorial libraries based on theinitial screen as well as an Env sequence alignment were sorted by thesame method. This resulted in BG505 SOSIP-MD39 which contained 11mutations and had improved expression and thermostability whilemaintaining a native-like antigenic profile. V3 mAb SPR was measuredwith IgG as the ligand and trimer as the analyte to allow avidity formaximum sensitivity, whereas bnAb Kds were measured as monovalentinteractions. These stabilizing mutations were combined with PGT121germline-targeting mutations to make native-like trimers with enhancedstability that engage PGT121 germline precursors.

FIG. 5 depicts postulated sequences of boosting immunogens to guidematuration along different paths following germline activation.

FIG. 6A-6B depicts a mammalian display-directed evolution design pathwayfor PGT121 germline-targeting Env-based immunogens. (FIG. 6A) Models ofthe PGT121 epitope are shown for each immunogen, with positions ofgermline-targeting mutations colored red and glycans depicted with cyanspheres. The epitope of BG505 is colored yellow (variable loop 1) andpink (variable loop 3). The paratope of PGT122 is mapped onto theepitope of BG505 and shown in tube representation (heavy chain in blue,light chain purple) (FIG. 6B) Binding KDs of mature, intermediatelymutated and germline-reverted variants of PGT121 for BG505-gp120 andgermline-targeting gp120s, determined by SPR. SPR K_(D)s are the averageof two or three experiments. *Indicates complex binding kinetics. WB,weak binding; (−), not done.

FIG. 7A-7C depicts a design of mutations to stabilize BG505-SOSIP andgermline-targeting native-like trimers. (FIG. 7A) Biophysical propertiesof stabilized BG505-SOSIP and germline-targeting trimers. Antigenicprofile was assessed by SPR, thermostability measurements were made byDSC, and expression was determined as yield of purified protein relativeto BG505-SOSIP made with PEI or 293Fectin transfection reagents in 293Fcells. Monovalent KDs were measured by SPR with trimer ligand and Fabanalyte except for PGT145 and PGDM1400, for which monovalent KDs weredetermined with IgG ligand and trimer analyte. For PGT151, a 1-to-1binding model gave a relatively poor kinetic fit. (FIG. 7B) Antigenicprofile of stabilized BG505-SOSIP and germline-targeting trimers byELISA. Data are representative of two independent experiments, each donein duplicate. (FIG. 7C) Mammalian display-directed evolution designpathways for engineering stabilized native-like trimers.

FIG. 8A-8F depicts sequential boosting schemes employing a native-liketrimer cocktail and germline-targeting design intermediates. (FIG. 8A)Side view of a single PGT122 Fab (light blue cartoon andsemi-transparent surface) bound to the BG505 SOSIP native-like gp140trimer, based on PDB ID: 4NCO. The PGT122-bound gp140 subunit is shownin wheat-colored cartoon; the V1, V2, V4, V5 variable loops on thatsubunit, modeled wherever missing in the crystal structure, are shown inyellow (V1), olive (V2), teal (V4), magenta (V5); the N332 glycan isshown as red spheres; and the two other gp140 subunits are shown as graysurfaces. (FIG. 8B) Same model as in (FIG. 8A), except thatglycosylation sites on the trimer have been decorated with Man8GlcNAc2glycans shown as spheres (V1 glycans, red; V2 glycans, olive; V4glycans, teal; N156 glycan, magenta; N332 glycan, red; all otherglycans, gray), and all trimer subunits are gray surface. (FIG. 8C)Two-dimensional histogram of variable loop (V1, V2, V4, V5) length andnumber of glycosylation sites among 3,897 unique HIV Env sequencesisolated from infected individuals obtained from www.hiv.lanl.gov.Frequency is indicated by the color scale shown for each loop. Thelength and number of glycosylation sites for each loop of thenative-like VLC trimers are indicated. Further elaboration of thiscocktail could include accounting for sequence variation atnon-variable-loop positions within the N332-epitope region (FIG. 14C).(FIG. 8D) Basic scheme in which a germline-targeting prime (10MUT or11MUT_(B)) is boosted by a native-like trimer (BG505) and then by acocktail of native-like trimers. (FIG. 8E) Diagram illustrating sevenboosting schemes employing germline-targeting design intermediates(7MUT, 6MUT, 5MUT, 3MUT) as boosts after a germline-targeting prime andbefore a native-like trimer; the scheme in A is included for reference.Relative affinity drops (in fold affinity decrease) for each boost,computed from FIG. 6B as described in the text, are indicated as rednumbers. (FIG. 8F) Linear diagrams of three of the best boosting schemesas ranked by favoring those with the smallest maximum affinity drop.

FIG. 9A-C depicts mammalian display overview related to FIG. 6. (FIG.9A) Schematic of mammalian display procedure. (FIG. 9B) Example FACSplots for unsorted mammalian display library (top) and the same librarysorted 3 times (bottom), see Extended Experimental Procedures. (FIG. 9C)Sequences of BG505-SOSIP (SEQ ID NO: 117) and BG505-gp120 (SEQ ID NO:118) used for mammalian display. Leader peptides are shown in red, thecMyc epitope is shown in green and the PDGFR TM is shown in blue.

FIG. 10 depicts germline-reverted PGT121 Abs, related to FIG. 6.Sequences of PGT121 and germline reverted variants with mutationshighlighted relative to germline V4-59/D3-3/J6 (heavy chain) andV3-21/J3 (light chain) genes. Figure discloses Heavy Chain sequences asSEQ ID NOS 119-128 and Light Chain sequences as SEQ ID NOS 129-138, allrespectively, in order of appearance.

FIG. 11 depicts PGT121 germline targeting gp120s and gp140s, related toFIG. 6. Designed germline targeting trimer and gp120 sequences are shownwith mutations from BG505-SOSIP highlighted. BG505-SOSIP.D664 containsthe mutation T332N, not highlighted. Figure discloses SEQ ID NOS139-152, respectively, in order of appearance.

FIG. 12A-12B depicts SPR binding data for V3 Abs binding to gp140 SOSIPsand their matching gp120s, related to FIG. 7. (FIG. 12A) Comparison ofV3 binding between WT BG505-SOSIP and BG505-SOSIP variants containingR304V and A319Y V3 mutations. For the 4025 SPR plots, the gp140concentrations tested are shown in M next to each relevant sensogram andare equivalent for all V3 Abs tested. (FIG. 12B) Comparison of V3binding to WT gp120 and gp120-MD16 containing the R304V and A319Ymutations. For the 4025 SPR plots, the gp120 concentrations tested areshown in M next to each relevant sensogram and are equivalent for all V3Abs tested.

FIG. 13A-13C depicts trimers with improved thermostability, expression,or antigenic profile, related to FIG. 7. (FIG. 13A) Sequences ofdesigned stabilized native-like trimers with mutations from BG505SOSIP.D664 highlighted. Figure discloses SEQ ID NOS 153-158,respectively, in order of appearance. (FIG. 13B) Melting temperature ofstabilized trimers as assessed by DSC. (FIG. 13C) Native-like trimerswith and without stabilizing mutations were transiently transfected in293F cells and expression levels were determined by capture ELISA usingPGT145 Fab for immobilization and PGT151 IgG for detection. Values arethe mean±SD of 3 replicate transfections.

FIG. 14A-14C depicts sequences and biophysical properties of theMD39-based VLC native-like trimer cocktail, and N332-epitope sequencediversity. (FIG. 14A) Biophysical characterization of the MD39-based VLCcocktail. (FIG. 14B) Sequences of MD39-based VLC cocktail members, withchanges relative to MD39 highlighted in green. Figure discloses SEQ IDNOS 159-163, respectively, in order of appearance. (FIG. 14C) List ofinterface positions on the BG505 SOSIP trimer near N332-supersite bnAbepitopes (PGT122, PGT128, PGT135), showing the frequencies of the aminoacids found at those positions in 10% or more of 3,897 unique HIV Envsequences isolated from infected individuals obtained fromwww.hiv.lanl.gov.

FIG. 15A-15G depicts properties of BG505 Olio6, Olio6 CD4KO, MD39 andMD37. FIG. 15A. Binding dissociation constants (KDs) measured by SPR forBG505 SOSIP, BG505 MD39 and BG505 Olio6 binding to various bnAbs. KDswere measured using trimer analyte and IgG captured on the sensor asligand for V1V2 apex antibodies that bind as one Fab per trimer, but forother antibodies the KDs were measured with trimer captured as ligandand Fabs as analytes. FIG. 15B. Binding of the native-like trimers BG505SOSIP and BG505 Olio6 and the non-native trimer BG505 gp120 foldon tomultiple non-nAbs, assessed by SPR. Trimers were analytes and IgG werecaptured on the sensor chip as ligand. Binding was assessed at 1 uMtrimer analyte concentration and is displayed as the ratio of theobserved response units to the response units expected for a 1 to 1binding interaction between each gp120 on the trimer and each Fab on theIgG. Lower values correspond to less binding. FIG. 15C. Binding of thenative-like trimers BG505 SOSIP, BG505 MD39, BG505 Olio6 and BG505 Olio6CD4KO, and the non-native trimer BG505 gp120 foldon, to various bnAbsassessed by ELISA and displayed as the antibody concentration that givesa half-maximal response (EC50). FIG. 15D. Binding of the native-liketrimers BG505 SOSIP, BG505 MD39, BG505 Olio6 and BG505 MD37 to variousbnAbs by ELISA and displayed as the area under the curve (AUC). FIG.15E. Binding of BG505 Olio6 and BG505 Olio6 CD4KO to CD4-IgG assessed bySPR. CD4-IgG was captured on the sensor as ligand and trimers wereanalytes. No binding was detected for BG505 Olio6 CD4KO at a maximumtrimer concentration of 1 uM. FIG. 15F. Binding of BG505 SOSIP and BG505Olio6 CD4KO to human CD4+ T cells by flow cytometry. Trimers werelabelled with two different fluorescent probes, cells were stained withtrimers for 30 mins, and trimer-specific binding was detected as thepercentage of cells in the double-positive population. 99.1% of cellsbound to BG505 SOSIP but only 0.1% of cells bound to BG505 Olio6 CD4KO.FIG. 15G. Size exclusion chromatography coupled inline with multi-anglelight scattering (SECMALS) analysis showing as examples that, insolution, BG505 MD39, BG505 Olio6, BG505 MD39c, and BG505 MD39 CP1.2GRSF4 have the molecular weight expected of a trimer.

FIG. 16A-16C depicts biophysical properties of stabilized trimers forBG505 and other HIV strains. FIG. 16A. Binding dissociation constants(KDs) measured by SPR for multiple stabilized trimers binding todifferent bnAbs (PGDM1400, PGT145, PGT121, PGT128, VRC01, 12A12, 3BNC60and PGT151). Also, the last column shows the melting temperature (TM)measured by differential scanning calorimetry (DSC). FIG. 16B. DSCmelting data for BG505 MD37, BG505 MD39, BG505 Olio6 and BG505 MD39C.FIG. 16C. DSC melting data for BG505 MD53, BG505 MD39 CP1.1, BG505 MD39CP1.2 and BG505 MD39 link14.

FIG. 17A-17C depicts properties of BG505 MD39 GRSF4. FIG. 17A. Side andBottom views of a model of BG505 MD39 GRSF4, illustrating the locationsof the five newly introduced glycosylation sites at positions 80, 241,289, 657 and 665. Addition of these glycosylation sites is intended tomask an otherwise exposed surface on the soluble BG505 trimer, a surfacethat is not targeted by the classes of bnAbs Applicants aim to elicitagainst the CD4bs, V2 apex and N332 supersite. Positions 241 and 289 areglycosylation sites that are relatively conserved across HIV strains butare absent in BG505. Positions 80 and 657 are not glycosylation sites in2869 HIV Env sequences Applicants examined, and 665 appears as aglycosylation site in 2 of 2869 sequences. FIG. 17B. Binding of thenative-like trimer BG505 MD39 GRSF4 to various bnAbs and the non-nAbs17b, 3074, 4025 and B6, assessed by SPR. Trimers were analytes and IgGwere captured on the sensor chip as ligand. Binding was assessed at 1 uMtrimer analyte concentration and is displayed as the ratio of theobserved response units to the response units expected for a 1 to 1binding interaction between each gp120 on the trimer and each Fab on theIgG. Lower values correspond to less binding. FIG. 17C. Dataillustrating that the additional glycans present in MD39 GRSF4 serve toblock undesired responses to soluble trimers. ELISA area under the curve(AUC) values are displayed for six VRC01 gH mice from Applicants' studyin Briney et al. Cell 2016. These six mice received four immunizations:eOD-GT8 60mer, core N276D 60mer, and two shots of BG505 SOSIP N276D, andseveral of them developed VRCO1-class responses to the CD4 binding site.Serum from these mice were tested by ELISA for the capacity to bind totwo variants of BG505 MD39 that each have mutations in the VRC01 epitopethat abrogate VRC01-class binding (“KO4” mutations). Thus serum bindingto these two variants report on the strength of off-target,non-VRCO1-class responses. This ELISA analysis shows that the sera from4/6 mice show considerably stronger reactivity to BG505 MD39 N276D KO4compared to BG505 MD39 GRSF4 KO4, demonstrating that a significantportion of the off-target response in these mice is directed to epitopesin the region masked by the additional GRSF4 glycans.

FIG. 18A-18D depicts properties of several cleavage-independent trimers.FIG. 18A. Antigenic profile of the cleavage-independent native-liketrimer BG505 CP1.2 GRSF4 compared to the cleavage-depdendent native-liketrimer BG505 MD39 and the non-native trimer BG505 gp120 foldon. Bindingwas assessed by SPR to various bnAbs and the non-nAbs 2557, 3074, 4025,F425, B6 and 17B. Trimers were analytes and IgG were captured on thesensor chip as ligand. Binding was assessed at 1 uM trimer analyteconcentration and is displayed as the ratio of the observed responseunits to the response units expected for a 1 to 1 binding interactionbetween each gp120 on the trimer and each Fab on the IgG. Lower valuescorrespond to less binding. FIG. 18B. Antigenic profiles of variouscleavage-independent trimers compared to the cleavage-dependent trimersBG505 SOSIP, BG505 MD39 and BG505 MD39 congly, measured as in A. FIG.18C. Expression levels of BG505 MD39 and various cleavage-independenttrimers. Applicants reported in Steichen et al. that BG505 MD39 has asignificantly higher expression level than BG505 SOSIP. FIG. 18D. Dataillustrating that the circular permutation modification combined withthe additional glycans present in MD39 CP1.2 GRSF4 serve to blockundesired responses induced by soluble trimers. ELISA titrations areshown for a single VRC01 gH mice that received four immunizations:eOD-GT8 60mer, core N276D 60mer, and two shots of BG505 MD39 N276D. Themouse serum binds equally well to BG505 MD39 N276D (which has a nativeCD4 binding site and VRC01 epitope except for the elimination of theN276 glycan which improves VRC01-class binding) and BG505 MD39 VRC01KO4(which has mutations in the VRC01 epitope that abrogate VRCO1-classbinding). Thus, the dominant serum response in this mouse is focused tooff-target, non-VRCO10-class epitopes on the trimer. The serum showsvery little reactivity to BG505 MD39 CP1.2 GRSF4, demonstrating that themodifications in this protein serve to block the majority of off-targetresponses in this mouse.

FIGS. 19A-19D depicts properties of the nanoparticle BG505 MD39 2JD6, an8-mer of MD39 trimers based on the ferritin from Pyrococcus Furiousis.FIG. 19A. SECMALS trace illustrating that the purified particle insolution has the molecular weight expected of a particle composed ofeight MD39 trimers each genetically fused via linkers to the 24-merferritin. FIG. 19B. Negative stain electron microscopy (EM) of BG505MD39 2JD6, illustrating the existence of ordered particles. FIG. 19C.Negative stain electron microscopy (EM) of BG505 MD39 2JD6, illustratingthe existence of ordered particles, at lower magnification and depictinga different sample field than in B. FIG. 19D. Antigenic profile of BG505MD39 2JD6, assessed by SPR. BG505 MD39 2JD6 particles were captured onthe sensor surface and Fabs of different antibodies were used asanalytes to assess monovalent binding interactions. The SPR kinetictraces and the measured dissociation constants (KDs) for four bnAbs areshown on the left. SPR kinetics for two non-nAbs are shown at right, andin those cases the binding signal was significantly weaker than for thebnAbs (note the different scale on the y-axis).

FIGS. 20A-20D depicts properties of the nanoparticle BG505 MD39 E2p, a20-mer of MD39 trimers based on the Dihydrolipoyl Transacetylase (E2p)from Bacillus Stearothermophilus. FIG. 20A. SECMALS trace illustratingthat the purified particle in solution has the molecular weight expectedof a particle composed of 20 MD39 trimers each genetically fused vialinkers to the underlying E2p 60-mer. FIG. 20B. Negative stain electronmicroscopy (EM) of BG505 MD39 E2p, illustrating the existence of orderedparticles. FIG. 20C. Negative stain electron microscopy (EM) of BG505MD39 E2p, illustrating the existence of ordered particles, at lowermagnification and depicting a different sample field than in B. FIG.20D. Antigenic profile of BG505 MD39 E2p, assessed by SPR. BG505 MD39E2p particles were captured on the sensor surface and Fabs of differentantibodies were used as analytes to assess monovalent bindinginteractions. The SPR kinetic traces and the measured dissociationconstants (KDs) for five bnAbs are shown on the left. SPR kinetics fortwo non-nAbs are shown at right, and in those cases the binding signalwas significantly weaker than for the bnAbs (note the different scale onthe y-axis).

FIG. 21A-21B depict flow cytometry data demonstrating cell-surfaceexpression and bnAb-binding of membrane-bound native-like trimers. FIG.21A. Binding of bnAbs to membrane-bound stabilized trimers using nativetransmembrane domains. FIG. 21B. Binding of bnAbs to membrane-boundstabilized trimers anchored to the membrane by flexible linkers andPDGFR transmembrane domains.

FIGS. 22A-22F depict results of NHP immunizations with BG505 Olio6 andBG505 Olio6 CD4-KO compared to BG505 SOSIP and two other comparatortrimers (BG505 SOSIP v4.1 and BG505 SOSIP v5.2). The sequence andbiophysical properties of BG505 SOSIP.v4.1 were reported in De Taeye etal. Cell 2015. The sequence and biophysical properties of BG505SOSIP.v5.2 are not known. Rhesus macaques (6 animals per immunogengroup) were immunized at 3 time points: week 0, week 8, and week 24. Allimmunizations were administered as split doses. Each immunizationconsisted of 2 subcutaneous injections of 50 μg of Env trimerprotein+37.5 units (U) of Iscomatrix adjuvant (CSL) in sterilephosphate-buffered saline (PBS) diluent for a total of 100 μg of Envtrimer protein+75 U of Iscomatrix per immunization per animal.Subcutaneous immunizations were given in a volume of 0.5 mL with a 1inch, 25 gauge needle at the medial inner mid-thigh of each leg.Throughout the figure immunogen names are abbreviated as follows: BG505SOSIP.664 (0.664 or BG505 WT), BG505 SOSIP.v4.1 (v4.1), BG505 SOSIP.v5.2(v5.2), BG505 SOSIP Olio6 (Olio6), and BG505 SOSIP Olio6 CD4-KO (Olio6CD4-KO). All nAb titer and ELISA binding Ab data panels show geometricmean titers with geometric SD. ns, non-significant. Also see FIG. 23.FIG. 22A. BG505 V3 loop peptide binding IgG titers in BG505 SOSIP.664,BG505 SOSIP.v4.1, BG505 SOSIP.v5.2, BG505 Olio6, and BG505 Olio6 CD4-KOimmunized RMs two weeks after the 3^(rd) immunization. Olio6 and Olio6CD4-KO both elicited significantly lower V3 binding antibody responsesthan BG505 SOSIP, indicating that the Olio6 and Olio6 CD4-KO retainednative structure in vivo to a greater than BG505 SOSIP. Horizontaldotted line indicates limit of detection. N=6 or 12. FIG. 22B. BG505SOSIP binding IgG titers in BG505 SOSIP.664, BG505 SOSIP.v4.1, BG505SOSIP.v5.2, BG505 Olio6, and BG505 Olio6 CD4-KO immunized RMs two weeksafter the 3′ immunization. Olio6 and Olio6 CD4-KO both elicitedsignificantly lower BG505 SOSIP-binding antibody responses than BG505SOSIP. Horizontal dotted line indicates limit of detection. Week 26, N=6or 12. FIG. 22C. Ratio of BG505 SOSIP to BG505 V3-peptide titers (asshown panels B and C) in immunized RMs two weeks after the 3^(rd)immunization. Olio6 and Olio6 CD4-KO both elicited responses with asignificantly higher ratio than BG505 SOSIP, reflecting the strongsuppression of V3 responses by Olio6 and Olio6 CD4-KO. Week 26, N=6 or12. FIG. 22D. Tier 1 SF162 nAb titers in BG505 SOSIP.664, BG505SOSIP.v4.1, BG505 SOSIP.v5.2, BG505 Olio6, and BG505 Olio6 CD4-KOimmunized animals two weeks after the 3^(rd) immunization. Olio6 andOlio6 CD4-KO both elicited significantly lower SF162 tier 1neutralization responses than BG505 SOSIP, also indicating that Olio6and Olio6 CD4-KO retained native structure in vivo to a greater thanBG505 SOSIP. Week 26, N=6 or 12. FIG. 22E. BG505 nAb titers in BG505SOSIP.664, BG505 SOSIP.v4.1, BG505 SOSIP.v5.2, BG505 Olio6, and BG505Olio6 CD4-KO immunized animals two weeks after the 3^(rd) immunization.Week 26, N=6 or 12. FIG. 22F. Ratio of BG505 and SF162 nAb titers (asshown in panels E and F) in immunized RMs two weeks after the 3^(rd)immunization. Olio6 and Olio6 CD4-KO both elicited responses with asignificantly higher ratio than BG505 SOSIP, further indicating thatOlio6 and Olio6 CD4-KO retained native structure in vivo to a greaterthan BG505 SOSIP. Week 26, N=6 or 12.

FIGS. 23A-23D depict additional results of NHP immunizations with BG505Olio6 and BG505 Olio6 CD4-KO compared to BG505 SOSIP and two othercomparator trimers (BG505 SOSIP v4.1 and BG505 SOSIP v5.2). The data inthis figure pertain to the same RM immunization experiment described inFIG. 22. Throughout the figure immunogen names are abbreviated asfollows: BG505 SOSIP.664 (0.664), BG505 SOSIP.v4.1 (v4.1), BG505SOSIP.v5.2 (v5.2), BG505 SOSIP Olio6 (Olio6), and BG505 SOSIP Olio6CD4-KO (Olio6 CD4-KO). ns, non-significant. All nAb titer and ELISAbinding Ab data panels show geometric mean titers with geometric SD.FIG. 23A. BG505 gp120 binding IgG titer 2 weeks after the 3^(rd)immunization. Olio6 and Olio6 CD4-KO both elicited significantly lowergp120 antibody binding responses than BG505 SOSIP, further indicatingthat Olio6 and Olio6 CD4-KO retained native structure in vivo to agreater than BG505 SOSIP. Week 26, N=6 or 12. FIG. 23B. Comparison ofV3-peptide ELISA titers against the BG505 Olio6 V3 loop peptide (Olio6)and the BG505 SOSIP.664 V3 loop peptide (WT). V3 loop peptide bindingIgG titers for both of these peptides were measured from plasma of RMsimmunized with BG505 SOSIP.664, BG505 Olio6, or BG505 Olio6 CD4-KO, twoweeks after the 3^(rd) immunization. Olio6 and Olio6 CD4-KO bothelicited significantly lower V3 binding antibody responses than BG505SOSIP, regardless of whether the ELISA measurement is performed with WTor Olio6 V3 peptides. Week 26, N=6. FIG. 23C. The ratio of BG505 nAbtiter to V3-peptide binding IgG titer after the 3^(rd) immunization.Week 26, N=6 or 12. FIG. 23D. Tier 1 MW965 nAb titers after the 3^(rd)immunization. Week 26, N=6 or 12.

FIGS. 24A-24B depict properties of BG505_MD39_CP1.2_GRSF7_qLoops1. FIG.24A. SECMALS analysis showing that the molecule is a trimer in solution.FIG. 24B. SPR analysis showing that the trimer exhibits a native-likeantigenic profile. In particular the trimer binds well to thetrimer-structure-specific bnAb PGT145, as well as two other bnAbs VRC01and PGT121, but does not exhibit significant affinity fornon-neutralizing Abs B6 or 4025.

DETAILED DESCRIPTION OF THE INVENTION

The invention pertains to the identification, design, synthesis andisolation of mutant trimers disclosed herein as well as nucleic acidsencoding the same. The present invention also relates to homologues,derivatives and variants of the sequences of the mutant trimers andnucleic acids encoding the same, wherein it is preferred that thehomologue, derivative or variant have at least 50%, at least 60%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 93%, at least 95%, at least 97%, at least 98% or at least 99%homology or identity with the sequence of the mutant trimers and nucleicacids encoding the same. It is noted that within this specification,homology to sequences of the mutant proteins and nucleic acids encodingthe same refers to the homology of the homologue, derivative or variantto the binding site of the mutant proteins and nucleic acids encodingthe same.

The invention still further relates to nucleic acid sequences expressingthe mutant trimers disclosed herein, or homologues, variants orderivatives thereof. One of skill in the art will know, recognize andunderstand techniques used to create such. Additionally, one of skill inthe art will be able to incorporate such a nucleic acid sequence into anappropriate vector, allowing for production of the amino acid sequenceof mutant proteins and nucleic acids encoding the same or a homologue,variant or derivative thereof.

Where used herein and unless specifically indicated otherwise, thefollowing terms are intended to have the following meanings in additionto any broader (or narrower) meanings the terms might enjoy in the art:

The term “isolated” or “non-naturally occurring” is used herein toindicate that the isolated moiety (e.g. peptide or compound) exists in aphysical milieu distinct from that in which it occurs in nature. Forexample, the isolated peptide may be substantially isolated with respectto the complex cellular milieu in which it naturally occurs. Theabsolute level of purity is not critical, and those skilled in the artmay readily determine appropriate levels of purity according to the useto which the peptide is to be put. The term “isolating” when used a stepin a process is to be interpreted accordingly.

In many circumstances, the isolated moiety will form part of acomposition (for example a more or less crude extract containing manyother molecules and substances), buffer system, matrix or excipient,which may for example contain other components (including proteins, suchas albumin).

In other circumstances, the isolated moiety may be purified to essentialhomogeneity, for example as determined by PAGE or column chromatography(for example HPLC or mass spectrometry). In preferred embodiments, theisolated peptide or nucleic acid of the invention is essentially thesole peptide or nucleic acid in a given composition.

In an advantageous embodiment, a tag may be utilized for purification orbiotinylation. The tag for purification may be a his tag. In anotherembodiment, the tag for biotinylation may be an avi-tag. Other tags arecontemplated for purification, however, purification may be accomplishedwithout a tag. In another embodiment, antibody (such as, not limited to,a broadly neutralizing antibody) affinity columns are contemplated. Inanother embodiment, lectin columns are contemplated.

Native-like soluble trimerscan be made by several methods that allinvolve stabilizing associations between envelope protein subunits. See,e.g., P. Dosenovic et al., “Immunization for HIV-1 broadly neutralizingantibodies in human Ig knockin mice,” Cell, 161:1-11, 2015; J. G.Jardine et al., “Priming a broadly neutralizing antibody response toHIV-1 using a germline targeting immunogen,” Science,doi:10.1126/science.aac5894, 2015 and R. W. Sanders et al., “HIV-1neutralalizing antibodies induced by native-like envelope trimers,”Science, doi: 10.1126/science.aac4223, 2015.

The proteins and compounds of the invention need not be isolated in thesense defined above, however.

The term “pharmaceutical composition” is used herein to define a solidor liquid composition in a form, concentration and level of puritysuitable for administration to a patient (e.g. a human patient) uponwhich administration it may elicit the desired physiological changes.The terms “immunogenic composition” and “immunological composition” and“immunogenic or immunological composition” cover any composition thatelicits an immune response against the targeted pathogen, HIV. Termssuch as “vaccinal composition” and “vaccine” and “vaccine composition”cover any composition that induces a protective immune response againstthe targeted pathogen or which efficaciously protects against thepathogen; for instance, after administration or injection, elicits aprotective immune response against the targeted pathogen or providesefficacious protection against the pathogen. Accordingly, an immunogenicor immunological composition induces an immune response, which may, butneed not, be a protective immune response. An immunogenic orimmunological composition may be used in the treatment of individualsinfected with the pathogen, e.g., to stimulate an immune responseagainst the pathogen, such as by stimulating antibodies against thepathogen. Thus, an immunogenic or immunological composition may be apharmaceutical composition. Furthermore, when the text speaks of“immunogen, antigen or epitope”, an immunogen may be an antigen or anepitope of an antigen. A diagnostic composition is a compositioncontaining a compound or antibody, e.g., a labeled compound or antibody,that is used for detecting the presence in a sample, such as abiological sample, e.g., blood, semen, vaginal fluid, etc, of anantibody that binds to the compound or an immunogen, antigen or epitopethat binds to the antibody; for instance, an anti-HIV antibody or an HIVimmunogen, antigen or epitope.

A “conservative amino acid change” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g. lysine, arginine and histidine), acidic side chains(e.g. aspartic acid and glutamic acid), non-charged amino acids or polarside chains (e.g. glycine, asparagine, glutamine, serine, threonine,tyrosine and cysteine), non-polar side chains (e.g. alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine and tryptophan),beta-branched side chains (e.g. threonine, valine and isoleucine), andaromatic side chains (e.g. tyrosine, phenylalanine, tryptophan andhistidine).

The terms “protein”, “peptide”, “polypeptide”, and “amino acid sequence”are used interchangeably herein to refer to polymers of amino acidresidues of any length. The polymer may be linear or branched, it maycomprise modified amino acids or amino acid analogs, and it may beinterrupted by chemical moieties other than amino acids. The terms alsoencompass an amino acid polymer that has been modified naturally or byintervention; for example disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling or bioactivecomponent.

As used herein, the terms “antigen” or “immunogen” are usedinterchangeably to refer to a substance, typically a protein, which iscapable of inducing an immune response in a subject. The term alsorefers to proteins that are immunologically active in the sense thatonce administered to a subject (either directly or by administering tothe subject a nucleotide sequence or vector that encodes the protein) isable to evoke an immune response of the humoral and/or cellular typedirected against that protein.

The term “antibody” includes intact molecules as well as fragmentsthereof, such as Fab, F(ab′)₂, Fv and scFv which are capable of bindingthe epitope determinant. These antibody fragments retain some ability toselectively bind with its antigen or receptor and include, for example:

(a) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule may be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(b) Fab′, the fragment of an antibody molecule may be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(c) F(ab′)₂, the fragment of the antibody that may be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(d) scFv, including a genetically engineered fragment containing thevariable region of a heavy and a light chain as a fused single chainmolecule.

General methods of making these fragments are known in the art. (See forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1988), which is incorporated herein byreference). Fabs, Fv and scFV may also be made recombinantly, i.e.expressed as Fab, Fv or scFV rather than cleaving an intact IgG.

A “neutralizing antibody” may inhibit the entry of HIV-1 virus forexample SF162 and/or JR-CSF with a neutralization index >1.5 or >2.0.Broad and potent neutralizing antibodies may neutralize greater thanabout 50% of HIV-1 viruses (from diverse clades and different strainswithin a clade) in a neutralization assay. The inhibitory concentrationof the monoclonal antibody may be less than about 25 mg/ml to neutralizeabout 50% of the input virus in the neutralization assay.

An “isolated antibody” or “non-naturally occurring antibody” is one thathas been separated and/or recovered from a component of its naturalenvironment. Contaminant components of its natural environment arematerials that would interfere with diagnostic or therapeutic uses forthe antibody, and may include enzymes, hormones, and other proteinaceousor nonproteinaceous solutes. In preferred embodiments, the antibody ispurified: (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight; (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator; or (3)to homogeneity by SDS-PAGE under reducing or non-reducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies which may comprise the population areidentical except for possible naturally occurring mutations that may bepresent in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigenic site. Furthermore, in contrastto polyclonal antibody preparations that include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Inaddition to their specificity, the monoclonal antibodies areadvantageous in that they may be synthesized uncontaminated by otherantibodies. The modifier “monoclonal” is not to be construed asrequiring production of the antibody by any particular method. Forexample, the monoclonal antibodies useful in the present invention maybe prepared by the hybridoma methodology first described by Kohler etal., Nature, 256:495 (1975), or may be made using recombinant DNAmethods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S.Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolatedfrom phage antibody libraries using the techniques described in Clacksonet al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.,222:581-597 (1991), for example.

An “antibody fragment” may comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂,scFV and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No.5,641,870; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]);single-chain antibody molecules; and multispecific antibodies formedfrom antibody fragments.

It should be understood that the proteins, including the antibodies ofthe invention may differ from the exact sequences illustrated anddescribed herein. Thus, the invention contemplates deletions, additionsand substitutions to the sequences shown, so long as the sequencesfunction in accordance with the methods of the invention. In thisregard, particularly preferred substitutions will generally beconservative in nature, i.e., those substitutions that take place withina family of amino acids. For example, amino acids are generally dividedinto four families: (1) acidic—aspartate and glutamate; (2)basic—lysine, arginine, histidine; (3) non-polar—alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and(4) uncharged polar—glycine, asparagine, glutamine, cysteine, serinethreonine, tyrosine. Phenylalanine, tryptophan, and tyrosine aresometimes classified as aromatic amino acids. It is reasonablypredictable that an isolated or non-naturally occurring replacement ofleucine with isoleucine or valine, or vice versa; an aspartate with aglutamate or vice versa; a threonine with a serine or vice versa; or asimilar conservative replacement of an amino acid with a structurallyrelated amino acid, will not have a major effect on the biologicalactivity. Proteins having substantially the same amino acid sequence asthe sequences illustrated and described but possessing minor amino acidsubstitutions that do not substantially affect the immunogenicity of theprotein are, therefore, within the scope of the invention.

As used herein the terms “nucleotide sequences” and “nucleic acidsequences” refer to deoxyribonucleic acid (DNA) or ribonucleic acid(RNA) sequences, including, without limitation, messenger RNA (mRNA),DNA/RNA hybrids, or synthetic nucleic acids. The nucleic acid may besingle-stranded, or partially or completely double-stranded (duplex).Duplex nucleic acids may be homoduplex or heteroduplex.

As used herein the term “transgene” may used to refer to “recombinant”nucleotide sequences that may be derived from any of the nucleotidesequences encoding the proteins of the present invention. The term“recombinant” means a nucleotide sequence that has been manipulated “byman” and which does not occur in nature, or is linked to anothernucleotide sequence or found in a different arrangement in nature. It isunderstood that manipulated “by man” means manipulated by someartificial means, including by use of machines, codon optimization,restriction enzymes, etc.

For example, in one embodiment the nucleotide sequences may be mutatedsuch that the activity of the encoded proteins in vivo is abrogated. Inanother embodiment the nucleotide sequences may be codon optimized, forexample the codons may be optimized for human use. In preferredembodiments the nucleotide sequences of the invention are both mutatedto abrogate the normal in vivo function of the encoded proteins, andcodon optimized for human use. For example, each of the sequences of theinvention, such as the mutant trimers, may be altered in these ways.

As regards codon optimization, the nucleic acid molecules of theinvention have a nucleotide sequence that encodes the antigens of theinvention and may be designed to employ codons that are used in thegenes of the subject in which the antigen is to be produced. Manyviruses, including HIV and other lentiviruses, use a large number ofrare codons and, by altering these codons to correspond to codonscommonly used in the desired subject, enhanced expression of theantigens may be achieved. In a preferred embodiment, the codons used are“humanized” codons, i.e., the codons are those that appear frequently inhighly expressed human genes (Andre et al., J. Virol. 72:1497-1503,1998) instead of those codons that are frequently used by HIV. Suchcodon usage provides for efficient expression of the transgenic HIVproteins in human cells. Any suitable method of codon optimization maybe used. Such methods, and the selection of such methods, are well knownto those of skill in the art. In addition, there are several companiesthat will optimize codons of sequences, such as Geneart (geneart.com).Thus, the nucleotide sequences of the invention may readily be codonoptimized.

The invention further encompasses nucleotide sequences encodingfunctionally and/or antigenically equivalent variants and derivatives ofthe antigens of the invention and functionally equivalent fragmentsthereof. These functionally equivalent variants, derivatives, andfragments display the ability to retain antigenic activity. Forinstance, changes in a DNA sequence that do not change the encoded aminoacid sequence, as well as those that result in conservativesubstitutions of amino acid residues, one or a few amino acid deletionsor additions, and substitution of amino acid residues by amino acidanalogs are those which will not significantly affect properties of theencoded polypeptide. Conservative amino acid substitutions areglycine/alanine; valine/isoleucine/leucine; asparagine/glutamine;aspartic acid/glutamic acid; serine/threonine/methionine;lysine/arginine; and phenylalanine/tyrosine/tryptophan. In oneembodiment, the variants have at least 50%, at least 55%, at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% homology oridentity to the antigen, epitope, immunogen, peptide or polypeptide ofinterest.

For the purposes of the present invention, sequence identity or homologyis determined by comparing the sequences when aligned so as to maximizeoverlap and identity while minimizing sequence gaps. In particular,sequence identity may be determined using any of a number ofmathematical algorithms. A nonlimiting example of a mathematicalalgorithm used for comparison of two sequences is the algorithm ofKarlin & Altschul, Proc. Natl. Acad. Sci. USA 1990; 87: 2264-2268,modified as in Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1993; 90:5873-5877.

Another example of a mathematical algorithm used for comparison ofsequences is the algorithm of Myers & Miller, CABIOS 1988; 4: 11-17.Such an algorithm is incorporated into the ALIGN program (version 2.0)which is part of the GCG sequence alignment software package. Whenutilizing the ALIGN program for comparing amino acid sequences, a PAM120weight residue table, a gap length penalty of 12, and a gap penalty of 4may be used. Yet another useful algorithm for identifying regions oflocal sequence similarity and alignment is the FASTA algorithm asdescribed in Pearson & Lipman, Proc. Natl. Acad. Sci. USA 1988; 85:2444-2448.

Advantageous for use according to the present invention is the WU-BLAST(Washington University BLAST) version 2.0 software. WU-BLAST version 2.0executable programs for several UNIX platforms may be downloaded fromftp://blast.wustl.edu/blast/executables. This program is based onWU-BLAST version 1.4, which in turn is based on the public domainNCBI-BLAST version 1.4 (Altschul & Gish, 1996, Local alignmentstatistics, Doolittle ed., Methods in Enzymology 266: 460-480; Altschulet al., Journal of Molecular Biology 1990; 215: 403-410; Gish & States,1993; Nature Genetics 3: 266-272; Karlin & Altschul, 1993; Proc. Natl.Acad. Sci. USA 90: 5873-5877; all of which are incorporated by referenceherein).

The various recombinant nucleotide sequences and antibodies of theinvention are made using standard recombinant DNA and cloningtechniques. Such techniques are well known to those of skill in the art.See for example, “Molecular Cloning: A Laboratory Manual”, secondedition (Sambrook et al. 1989).

The nucleotide sequences of the present invention may be inserted into“vectors.” The term “vector” is widely used and understood by those ofskill in the art, and as used herein the term “vector” is usedconsistent with its meaning to those of skill in the art. For example,the term “vector” is commonly used by those skilled in the art to referto a vehicle that allows or facilitates the transfer of nucleic acidmolecules from one environment to another or that allows or facilitatesthe manipulation of a nucleic acid molecule.

Any vector that allows expression of the antibodies of the presentinvention may be used in accordance with the present invention. Incertain embodiments, the antibodies of the present invention may be usedin vitro (such as using cell-free expression systems) and/or in culturedcells grown in vitro in order to produce the encoded HIV− antibodies,which may then be used for various applications such as in theproduction of proteinaceous vaccines. For such applications, any vectorthat allows expression of the antibodies in vitro and/or in culturedcells may be used.

For applications where it is desired that the antibodies be expressed invivo, for example when the transgenes of the invention are used in DNAor DNA-containing vaccines, any vector that allows for the expression ofthe antibodies of the present invention and is safe for use in vivo maybe used. In preferred embodiments the vectors used are safe for use inhumans, mammals and/or laboratory animals.

For the antibodies of the present invention to be expressed, the proteincoding sequence should be “operably linked” to regulatory or nucleicacid control sequences that direct transcription and translation of theprotein. As used herein, a coding sequence and a nucleic acid controlsequence or promoter are said to be “operably linked” when they arecovalently linked in such a way as to place the expression ortranscription and/or translation of the coding sequence under theinfluence or control of the nucleic acid control sequence. The “nucleicacid control sequence” may be any nucleic acid element, such as, but notlimited to promoters, enhancers, IRES, introns, and other elementsdescribed herein that direct the expression of a nucleic acid sequenceor coding sequence that is operably linked thereto. The term “promoter”will be used herein to refer to a group of transcriptional controlmodules that are clustered around the initiation site for RNA polymeraseII and that when operationally linked to the protein coding sequences ofthe invention lead to the expression of the encoded protein. Theexpression of the transgenes of the present invention may be under thecontrol of a constitutive promoter or of an inducible promoter, whichinitiates transcription only when exposed to some particular externalstimulus, such as, without limitation, antibiotics such as tetracycline,hormones such as ecdysone, or heavy metals. The promoter may also bespecific to a particular cell-type, tissue or organ. Many suitablepromoters and enhancers are known in the art, and any such suitablepromoter or enhancer may be used for expression of the transgenes of theinvention. For example, suitable promoters and/or enhancers may beselected from the Eukaryotic Promoter Database (EPDB).

The vectors used in accordance with the present invention shouldtypically be chosen such that they contain a suitable gene regulatoryregion, such as a promoter or enhancer, such that the antibodies of theinvention may be expressed.

Any suitable vector may be used depending on the application. Forexample, plasmids, viral vectors, bacterial vectors, protozoal vectors,insect vectors, baculovirus expression vectors, yeast vectors, mammaliancell vectors, and the like, may be used. Suitable vectors may beselected by the skilled artisan taking into consideration thecharacteristics of the vector and the requirements for expressing theantibodies under the identified circumstances.

In an advantageous embodiment, IgG1 and Fab expression vectors may beutilized to reconstitute heavy and light chain constant regions if heavyand light chain genes of the antibodies of the present invention arecloned.

When the aim is to express the antibodies of the invention in vivo in asubject, for example in order to generate an immune response against anHIV-1 antigen and/or protective immunity against HIV-1, expressionvectors that are suitable for expression on that subject, and that aresafe for use in vivo, should be chosen. For example, in some embodimentsit may be desired to express the antibodies of the invention in alaboratory animal, such as for pre-clinical testing of the HIV-1immunogenic compositions and vaccines of the invention. In otherembodiments, it will be desirable to express the antibodies of theinvention in human subjects, such as in clinical trials and for actualclinical use of the immunogenic compositions and vaccine of theinvention. Any vectors that are suitable for such uses may be employed,and it is well within the capabilities of the skilled artisan to selecta suitable vector. In some embodiments it may be preferred that thevectors used for these in vivo applications are attenuated to vectorfrom amplifying in the subject. For example, if plasmid vectors areused, preferably they will lack an origin of replication that functionsin the subject so as to enhance safety for in vivo use in the subject.If viral vectors are used, preferably they are attenuated orreplication-defective in the subject, again, so as to enhance safety forin vivo use in the subject.

In preferred embodiments of the present invention viral vectors areused. Viral expression vectors are well known to those skilled in theart and include, for example, viruses such as adenoviruses,adeno-associated viruses (AAV), alphaviruses, herpesviruses,retroviruses and poxviruses, including avipox viruses, attenuatedpoxviruses, vaccinia viruses, and particularly, the modified vacciniaAnkara virus (MVA; ATCC Accession No. VR-1566). Such viruses, when usedas expression vectors are innately non-pathogenic in the selectedsubjects such as humans or have been modified to render themnon-pathogenic in the selected subjects. For example,replication-defective adenoviruses and alphaviruses are well known andmay be used as gene delivery vectors.

The nucleotide sequences and vectors of the invention may be deliveredto cells, for example if the aim is to express the HIV-1 antigens incells in order to produce and isolate the expressed proteins, such asfrom cells grown in culture. For expressing the antibodies in cells anysuitable transfection, transformation, or gene delivery methods may beused. Such methods are well known by those skilled in the art, and oneof skill in the art would readily be able to select a suitable methoddepending on the nature of the nucleotide sequences, vectors, and celltypes used. For example, transfection, transformation, microinjection,infection, electroporation, lipofection, or liposome-mediated deliverycould be used. Expression of the antibodies may be carried out in anysuitable type of host cells, such as bacterial cells, yeast, insectcells, and mammalian cells. The antibodies of the invention may also beexpressed using including in vitro transcription/translation systems.All of such methods are well known by those skilled in the art, and oneof skill in the art would readily be able to select a suitable methoddepending on the nature of the nucleotide sequences, vectors, and celltypes used.

A synthetic mutant trimer may be chemically synthesized in whole or partusing techniques that are well-known in the art (see, e.g.,Kochendoerfer, G. G., 2001). Additionally, homologs and derivatives ofthe polypeptide may be also be synthesized.

Alternatively, methods which are well known to those skilled in the artmay be used to construct expression vectors containing nucleic acidmolecules that encode the polypeptide or homologs or derivatives thereofunder appropriate transcriptional/translational control signals, forexpression. These methods include in vitro recombinant DNA techniques,synthetic techniques and in vivo recombination/genetic recombination.See, for example, the techniques described in Maniatis et al., 1989.

The HIV envelope protein (Env) is the target of broadly neutralizingantibodies (bnAbs) in natural infection. Env is a membrane proteincomposed of a trimer of gp120 and gp41 subunits that contains a highdegree of sequence diversity and a surface that is shielded by N-linkedglycans. The bnAbs that target Env often have unusual features such as along complementarity-determining region (CDR) H3, high levels of somatichypermutation (SHM), and insertions and deletions (INDELS). Furthermore,most of the bnAbs recognize complex epitopes that are typicallynon-linear and have both protein and glycan components.

The most common epitope of bnAbs in HIV infected individuals is a highmannose glycan patch at the base of the variable loop V3 that includes aglycan linked to N332 (Landais et al. 2016 PLoS Pathog. 12, e1005369).PGT121 and its somatic relatives are an exceptionally potent family ofbnAbs that target this epitope and PGT121 has been shown to protectmacaques in SHIV challenge studies (Walker et al. 2011 Nature. 477,466-470, Moldt et al. 2012 Proc Natl Acad Sci. 109, 18921-18925). Theelicitation of high and sustained titers of PGT121-like antibodies byvaccination would therefore have a reasonable likelihood of providingprotection against HIV in humans.

HIV Env proteins show no detectable affinity for predicted germlineprecursors of PGT121, suggesting that activation of appropriateprecursors is a barrier to PGT121-like bnAb induction that could beaddressed by germline-targeting immunogen design. In this view, vaccineinduction of PGT121-like bnAbs might be achieved by a germline-targetingprime followed by boosts with progressively more native-like Env,similar to what has been proposed for elicitation of VRC01-class bnAbs(Jardine, Julien, Menis et al. 2013 Science. 340, 711-716; McGuire et al2013 J Exp Med. 210, 655-663; Jardine, Ota, Sok et al. 2015 Science.349, 156-161). BG505 SOSIP.664 was the first soluble native-like Envtrimer (Sanders et al. 2013 PloS Pathog. 9, e1003618). In parallel withApplicants' germline-targeting effort a goal was to improve theexpression, stability and antigenic profile of BG505 SOSIP.664 in orderto have an enhanced trimer platform for germline targeting and boosting.Using a lentivirus-based method for displaying libraries of immunogenson the surface of mammalian cells, and guided by the known structure ofBG505 SOSIP.664 (Julien et al. 2013 Science. 342, 1477-1483; Lyumkis etal. 2013 Science. 342, 1484-1490; Pancera et al. 2014 Nature. 514,455-461) Applicants have engineered a series of soluble native-liketrimers with improved yield, thermostability and antigenic profile,which have progressively increasing affinity for putative PGT121germline precursors and intermediately mutated antibodies.

Applicants have demonstrated that structure-guided mammalian cellsurface display can be used to engineer trimers containing native-likeglycans. Native-like trimers have been developed that bind to predictedPGT121 germline precursors and intermediately mutated antibodies BG505SOSIP trimers were engineered with improved yield, thermostability andantigenic profile. Tests of priming and boosting strategies arecurrently underway in PGT121-GL knock-in mice.

Applicants claim sequences of different types of immunogen sequences.The sequences provided below are exemplary examples, the stabilizingmutations, modifications, (such as, but not limited to,cleavage-independent modifications), and/or a membrane anchoringstrategy (such as, but not limited to, linker plus platelet-derivedgrowth factor receptor (PDGFR)) described herein are applicable to anyHIV strain or clade, such as but not limited to, those described below.

As used herein, at least three separate zoonotic transmissions resultedin the formation of three distinct HIV-1 groups: M (main), O (outlier),and N (non-M/non-O).

About 90% of HIV-1 infections are classified as group M and these aredistributed worldwide. Group O infections are endemic to several westcentral African countries and represent 1 to 5% of all HIV-1 infectionin those areas. Group N has only been identified in a small number ofindividuals in Cameroon.

Within the HIV-M group, there is a further division into at least tensubtypes or clades (groups of genetically related virus). Historically,the distribution of subtypes followed the geographic patterns listedbelow.

Clade or Subtype A: Central and East Africa as well as East Europeancountries that were formerly part of the Soviet Union.

Clade or Subtype B: West and Central Europe, the Americas, Australia,South America, and several southeast Asian countries (Thailand, andJapan), as well as northern Africa and the Middle East.

Clade or Subtype C: Sub-Saharan Africa, India, and Brazil.

Clade or Subtype D: North Africa and the Middle East.

Clade or Subtype F: South and southeast Asia.

Clade or Subtype G: West and Central Africa.

Clade or Subtypes H, J, and K: Africa and the Middle East.

Additionally, different subtypes can combine genetic material to form ahybrid virus, known as a “circulating recombinant form” (CRFs), of whichat least twenty have been identified (see, e.g., 2. Buonarguro L HumanImmunodeficiency Virus Type 1 Subtype distribution in the worldwideepidemic: pathogenetic and therapeutic implications. J Virol81(19):10209-19, 2007).

The present invention encompasses the stabilizing mutations,modifications, (such as, but not limited to, cleavage-independentmodifications), and/or a membrane anchoring strategy (such as, but notlimited to, linker plus platelet-derived growth factor receptor (PDGFR))described herein to all groups and clades of HIV.

Types I and II are gp120 molecules (I) and gp140 trimer molecules (II)with mutations discovered to improve binding to germline-reverted and/orless-mutated versions of PGT121. The sequences in I and II can beemployed in sequential immunization schemes to attempt to elicitPGT121-class bnAbs against HIV.

Type III are gp140 trimer molecules with stabilizing mutations toincrease expression level and/or increase thermal melting temperatureand/or improve antigenic profile, where a favorable antigenic profilemeans better affinity for broadly neutralizing antibodies and no or veryweak affinity for non-neutralizing antibodies.

Type IV are combinations of mutations from II and III: these are gp140trimers that contain both stabilizing mutations and germline-targetingmutations. In type IV Applicants have listed only a few importantcombinations, but the present invention encompasses all possiblecombinations of the mutations from II and III.

Type V are trimers with modified surfaces or of different strains thanBG505, that can be employed in strategic boosting regimens.

Type VI are additional trimer modifications that add extra functionalityand that can be combined with types II, III, IV or V.

Type VII are examples of native-like trimers from other HIV strains thathave been stabilized by MD39 and Olio6 mutations, demonstrating thegeneral applicability of the MD39 and Olio6 stabilizing mutations. FIG.16A provides experimental data supporting these trimers as native-like.

Type VIII are variants of BG505 MD39 that do not require cleavage byfurin. We refer to these as “cleavage-independent” trimers.

Type IX are glycan masked trimers in which N-linked glycosylation siteshave been added to cover the bottom and sides of the soluble trimer.

Type X are native-like trimers with variable loops V1, V2b and V4modified to both minimize their lengths and maximize the number ofglycosylation sites contained within them.

Type XI are BG505 MD39-based, single-component, self-assemblingnanoparticles.

Type XII are BG505 MD39-based, membrane-bound native like trimers.

Amino acid and nucleic acid sequences are listed below. In the aminoacid sequences, mutations relative to a parent construct are generallyindicated in bold.

I: gp120s with PGT121-Class Germline-Targeting Mutations

BG505-gp120-L111A-2_T135A_T139I_mC (BG505-gp120 3mut) (SEQ ID NO: 1)VWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISAWDQSLKPCVKLTPLCVTLQCTNVANNIIDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKY KVVKIEP(SEQ ID NO: 2) GTGTGGAAAGACGCCGAGACTACTCTGTTCTGTGCCAGCGACGCCAAAGCATACGAAACTGAAAAGCATAATGTGTGGGCTACTCACGCCTGCGTGCCCACAGACCCAAATCCCCAGGAAATCCACCTGGAGAATGTCACTGAGGAATTCAACATGTGGAAGAACAATATGGTGGAGCAGATGCATACCGACATCATTTCAGCCTGGGATCAGAGCCTGAAGCCATGCGTGAAACTGACTCCCCTGTGCGTCACCCTGCAGTGTACCAACGTGGCCAACAATATCATCGACGATATGCGGGGCGAACTGAAGAATTGTTCTTTCAACATGACCACAGAGCTGCGGGACAAGAAACAGAAAGTGTACAGTCTGTTTTATAGACTGGATGTGGTCCAGATCAATGAAAACCAGGGGAATCGATCCAACAATTCTAACAAGGAGTACCGGCTGATCAATTGCAACACTAGCGCCATTACCCAGGCTTGTCCCAAAGTGTCCTTTGAACCTATCCCAATTCATTATTGCGCCCCTGCTGGCTTCGCCATCCTGAAGTGTAAAGATAAGAAGTTCAACGGCACCGGGCCCTGCCCTTCTGTGAGTACAGTCCAGTGTACTCACGGGATTAAGCCTGTGGTCAGTACACAGCTGCTGCTGAATGGATCACTGGCTGAGGAAGAAGTGATGATCCGATCCGAGAACATTACTAACAATGCAAAGAATATCCTGGTGCAGTTCAACACCCCTGTCCAGATTAATTGCACTCGCCCAAACAATAACACCCGGAAAAGCATCAGAATTGGACCAGGCCAGGCATTTTACGCCACCGGGGACATCATTGGAGATATCAGACAGGCACACTGTAATGTGTCCAAGGCCACCTGGAACGAAACACTGGGAAAGGTGGTCAAACAGCTGAGAAAACATTTCGGCAATAACACTATCATTAGGTTTGCTAATAGCTCCGGCGGGGACCTGGAAGTGACTACCCACAGCTTCAACTGCGGAGGCGAGTTCTTTTACTGTAACACATCCGGCCTGTTTAATTCTACATGGATCAGTAACACTTCAGTGCAGGGCTCAAATAGCACCGGGAGCAACGATTCCATCACACTGCCTTGCCGCATTAAGCAGATCATTAATATGTGGCAGCGAATTGGACAGGCTATGTATGCACCCCCTATCCAGGGCGTGATTAGATGTGTCTCTAATATCACCGGGCTGATTCTGACACGCGACGGGGGATCTACAAACAGTACAACTGAGACTTTCAGGCCAGGCGGGGGAGACATGAGGGATAACTGGCGCAGCGAACTGTACAAGTATAAAGTGGTCAAAAT CGAGCCCBG505-gp120-L111A-2_5mut_mC (BG505-gp120 5mut) (SEQ ID NO: 3)VWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISAWDQSLKPCVKLTPLCVTLQCTNYTPNLTNDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKY KVVKIEP(SEQ ID NO: 4) GTGTGGAAAGACGCCGAGACTACTCTGTTCTGTGCCAGCGACGCCAAAGCATACGAAACTGAAAAGCATAATGTGTGGGCTACTCACGCCTGCGTGCCCACAGACCCAAATCCCCAGGAAATCCACCTGGAGAATGTCACTGAGGAATTCAACATGTGGAAGAACAATATGGTGGAGCAGATGCATACCGACATCATTTCAGCCTGGGATCAGAGCCTGAAGCCATGCGTGAAACTGACTCCCCTGTGCGTCACCCTGCAGTGTACCAACTACACACCCAATCTGACCAACGATATGCGGGGCGAACTGAAGAATTGTTCTTTCAACATGACCACAGAGCTGCGGGACAAGAAACAGAAAGTGTACAGTCTGTTTTATAGACTGGATGTGGTCCAGATCAATGAAAACCAGGGGAATCGATCCAACAATTCTAACAAGGAGTACCGGCTGATCAATTGCAACACTAGCGCCATTACCCAGGCTTGTCCCAAAGTGTCCTTTGAACCTATCCCAATTCATTATTGCGCCCCTGCTGGCTTCGCCATCCTGAAGTGTAAAGATAAGAAGTTCAACGGCACCGGGCCCTGCCCTTCTGTGAGTACAGTCCAGTGTACTCACGGGATTAAGCCTGTGGTCAGTACACAGCTGCTGCTGAATGGATCACTGGCTGAGGAAGAAGTGATGATCCGATCCGAGAACATTACTAACAATGCAAAGAATATCCTGGTGCAGTTCAACACCCCTGTCCAGATTAATTGCACTCGCCCAAACAATAACACCCGGAAAAGCATCAGAATTGGACCAGGCCAGGCATTTTACGCCACCGGGGACATCATTGGAGATATCAGACAGGCACACTGTAATGTGTCCAAGGCCACCTGGAACGAAACACTGGGAAAGGTGGTCAAACAGCTGAGAAAACATTTCGGCAATAACACTATCATTAGGTTTGCTAATAGCTCCGGCGGGGACCTGGAAGTGACTACCCACAGCTTCAACTGCGGAGGCGAGTTCTTTTACTGTAACACATCCGGCCTGTTTAATTCTACATGGATCAGTAACACTTCAGTGCAGGGCTCAAATAGCACCGGGAGCAACGATTCCATCACACTGCCTTGCCGCATTAAGCAGATCATTAATATGTGGCAGCGAATTGGACAGGCTATGTATGCACCCCCTATCCAGGGCGTGATTAGATGTGTCTCTAATATCACCGGGCTGATTCTGACACGCGACGGGGGATCTACAAACAGTACAACTGAGACTTTCAGGCCAGGCGGGGGAGACATGAGGGATAACTGGCGCAGCGAACTGTACAAGTATAAAGTGGTCAAAAT CGAGCCCBG505-gp120-L111A-2_7mut_mC (BG505-gp120 7mut or BG505 gp120 7MUT) (SEQID NO: 5) VWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISAWDQSLKPCVKLTPLCVTLQCTNYAPNLINDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKY KVVKIEP(SEQ ID NO: 6) GTGTGGAAAGACGCCGAGACTACTCTGTTCTGTGCCAGCGACGCCAAAGCATACGAAACTGAAAAGCATAATGTGTGGGCTACTCACGCCTGCGTGCCCACAGACCCAAATCCCCAGGAAATCCACCTGGAGAATGTCACTGAGGAATTCAACATGTGGAAGAACAATATGGTGGAGCAGATGCATACCGACATCATTTCAGCCTGGGATCAGAGCCTGAAGCCATGCGTGAAACTGACTCCCCTGTGCGTCACCCTGCAGTGTACCAACTACGCCCCCAATCTGATCAACGATATGCGGGGCGAACTGAAGAATTGTTCTTTCAACATGACCACAGAGCTGCGGGACAAGAAACAGAAAGTGTACAGTCTGTTTTATAGACTGGATGTGGTCCAGATCAATGAAAACCAGGGGAATCGATCCAACAATTCTAACAAGGAGTACCGGCTGATCAATTGCAACACTAGCGCCATTACCCAGGCTTGTCCCAAAGTGTCCTTTGAACCTATCCCAATTCATTATTGCGCCCCTGCTGGCTTCGCCATCCTGAAGTGTAAAGATAAGAAGTTCAACGGCACCGGGCCCTGCCCTTCTGTGAGTACAGTCCAGTGTACTCACGGGATTAAGCCTGTGGTCAGTACACAGCTGCTGCTGAATGGATCACTGGCTGAGGAAGAAGTGATGATCCGATCCGAGAACATTACTAACAATGCAAAGAATATCCTGGTGCAGTTCAACACCCCTGTCCAGATTAATTGCACTCGCCCAAACAATAACACCCGGAAAAGCATCAGAATTGGACCAGGCCAGGCATTTTACGCCACCGGGGACATCATTGGAGATATCAGACAGGCACACTGTAATGTGTCCAAGGCCACCTGGAACGAAACACTGGGAAAGGTGGTCAAACAGCTGAGAAAACATTTCGGCAATAACACTATCATTAGGTTTGCTAATAGCTCCGGCGGGGACCTGGAAGTGACTACCCACAGCTTCAACTGCGGAGGCGAGTTCTTTTACTGTAACACATCCGGCCTGTTTAATTCTACATGGATCAGTAACACTTCAGTGCAGGGCTCAAATAGCACCGGGAGCAACGATTCCATCACACTGCCTTGCCGCATTAAGCAGATCATTAATATGTGGCAGCGAATTGGACAGGCTATGTATGCACCCCCTATCCAGGGCGTGATTAGATGTGTCTCTAATATCACCGGGCTGATTCTGACACGCGACGGGGGATCTACAAACAGTACAACTGAGACTTTCAGGCCAGGCGGGGGAGACATGAGGGATAACTGGCGCAGCGAACTGTACAAGTATAAAGTGGTCAAAAT CGAGCCCBG505-gp120-L111A-2_10mut_mC (BG505-gp120 10mut or BG505 gp120 10MUT)(SEQ ID NO: 7) VWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISAWDQSLKPCVKLTPLCVTLQCTNYAPFLINDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYAFGDIIGDIRMAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKY KVVKIEP(SEQ ID NO: 8) GTGTGGAAAGACGCCGAGACTACTCTGTTCTGTGCCAGCGACGCCAAAGCATACGAAACTGAAAAGCATAATGTGTGGGCTACTCACGCCTGCGTGCCCACAGACCCAAATCCCCAGGAAATCCACCTGGAGAATGTCACTGAGGAATTCAACATGTGGAAGAACAATATGGTGGAGCAGATGCATACCGACATCATTTCAGCCTGGGATCAGAGCCTGAAGCCATGCGTGAAACTGACTCCCCTGTGCGTCACCCTGCAGTGTACCAACTACGCCCCCTTCCTGATCAACGATATGCGGGGCGAACTGAAGAATTGTTCTTTCAACATGACCACAGAGCTGCGGGACAAGAAACAGAAAGTGTACAGTCTGTTTTATAGACTGGATGTGGTCCAGATCAATGAAAACCAGGGGAATCGATCCAACAATTCTAACAAGGAGTACCGGCTGATCAATTGCAACACTAGCGCCATTACCCAGGCTTGTCCCAAAGTGTCCTTTGAACCTATCCCAATTCATTATTGCGCCCCTGCTGGCTTCGCCATCCTGAAGTGTAAAGATAAGAAGTTCAACGGCACCGGGCCCTGCCCTTCTGTGAGTACAGTCCAGTGTACTCACGGGATTAAGCCTGTGGTCAGTACACAGCTGCTGCTGAATGGATCACTGGCTGAGGAAGAAGTGATGATCCGATCCGAGAACATTACTAACAATGCAAAGAATATCCTGGTGCAGTTCAACACCCCTGTCCAGATTAATTGCACTCGCCCAAACAATAACACCCGGAAAAGCATCAGAATTGGACCAGGCCAGGCATTTTACGCCTTCGGGGACATCATTGGAGATATCAGAATGGCACACTGTAATGTGTCCAAGGCCACCTGGAACGAAACACTGGGAAAGGTGGTCAAACAGCTGAGAAAACATTTCGGCAATAACACTATCATTAGGTTTGCTAATAGCTCCGGCGGGGACCTGGAAGTGACTACCCACAGCTTCAACTGCGGAGGCGAGTTCTTTTACTGTAACACATCCGGCCTGTTTAATTCTACATGGATCAGTAACACTTCAGTGCAGGGCTCAAATAGCACCGGGAGCAACGATTCCATCACACTGCCTTGCCGCATTAAGCAGATCATTAATATGTGGCAGCGAATTGGACAGGCTATGTATGCACCCCCTATCCAGGGCGTGATTAGATGTGTCTCTAATATCACCGGGCTGATTCTGACACGCGACGGGGGATCTACAAACAGTACAACTGAGACTTTCAGGCCAGGCGGGGGAGACATGAGGGATAACTGGCGCAGCGAACTGTACAAGTATAAAGTGGTCAAAAT CGAGCCCBG505-gp120-L111A-2_10mut2A_mC (SEQ ID NO: 9)VWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISAWDQSLKPCVKLTPLCVTLQCTNYAPFLINNMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYAFGDIIGDIRMAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKY KVVKIEP(SEQ ID NO: 10)GTGTGGAAAGACGCCGAGACTACTCTGTTCTGTGCCAGCGACGCCAAAGCATACGAAACTGAAAAGCATAATGTGTGGGCTACTCACGCCTGCGTGCCCACAGACCCAAATCCCCAGGAAATCCACCTGGAGAATGTCACTGAGGAATTCAACATGTGGAAGAACAATATGGTGGAGCAGATGCATACCGACATCATTTCAGCCTGGGATCAGAGCCTGAAGCCATGCGTGAAACTGACTCCCCTGTGCGTCACCCTGCAGTGTACCAACTACGCCCCCTTCCTGATCAACAACATGCGGGGCGAACTGAAGAATTGTTCTTTCAACATGACCACAGAGCTGCGGGACAAGAAACAGAAAGTGTACAGTCTGTTTTATAGACTGGATGTGGTCCAGATCAATGAAAACCAGGGGAATCGATCCAACAATTCTAACAAGGAGTACCGGCTGATCAATTGCAACACTAGCGCCATTACCCAGGCTTGTCCCAAAGTGTCCTTTGAACCTATCCCAATTCATTATTGCGCCCCTGCTGGCTTCGCCATCCTGAAGTGTAAAGATAAGAAGTTCAACGGCACCGGGCCCTGCCCTTCTGTGAGTACAGTCCAGTGTACTCACGGGATTAAGCCTGTGGTCAGTACACAGCTGCTGCTGAATGGATCACTGGCTGAGGAAGAAGTGATGATCCGATCCGAGAACATTACTAACAATGCAAAGAATATCCTGGTGCAGTTCAACACCCCTGTCCAGATTAATTGCACTCGCCCAAACAATAACACCCGGAAAAGCATCAGAATTGGACCAGGCCAGGCATTTTACGCCTTCGGGGACATCATTGGAGATATCAGAATGGCACACTGTAATGTGTCCAAGGCCACCTGGAACGAAACACTGGGAAAGGTGGTCAAACAGCTGAGAAAACATTTCGGCAATAACACTATCATTAGGTTTGCTAATAGCTCCGGCGGGGACCTGGAAGTGACTACCCACAGCTTCAACTGCGGAGGCGAGTTCTTTTACTGTAACACATCCGGCCTGTTTAATTCTACATGGATCAGTAACACTTCAGTGCAGGGCTCAAATAGCACCGGGAGCAACGATTCCATCACACTGCCTTGCCGCATTAAGCAGATCATTAATATGTGGCAGCGAATTGGACAGGCTATGTATGCACCCCCTATCCAGGGCGTGATTAGATGTGTCTCTAATATCACCGGGCTGATTCTGACACGCGACGGGGGATCTACAAACAGTACAACTGAGACTTTCAGGCCAGGCGGGGGAGACATGAGGGATAACTGGCGCAGCGAACTGTACAAGTATAAAGTGGTCAAAAT CGAGCCCBG505-gp120-L111A-2_11mut2A_mC (SEQ ID NO: 11)VWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISAWDQSLKPCVKLTPLCVTLQCTNYAPNLLSNMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYAFGDIIGDIRMAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSIVIPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKY KVVKIEP(SEQ ID NO: 12)GTGTGGAAAGACGCCGAGACTACTCTGTTCTGTGCCAGCGACGCCAAAGCATACGAAACTGAAAAGCATAATGTGTGGGCTACTCACGCCTGCGTGCCCACAGACCCAAATCCCCAGGAAATCCACCTGGAGAATGTCACTGAGGAATTCAACATGTGGAAGAACAATATGGTGGAGCAGATGCATACCGACATCATTTCAGCCTGGGATCAGAGCCTGAAGCCATGCGTGAAACTGACTCCCCTGTGCGTCACCCTGCAGTGTACCAACTACGCCCCCAACCTGCTGAGCAACATGCGGGGCGAACTGAAGAATTGTTCTTTCAACATGACCACAGAGCTGCGGGACAAGAAACAGAAAGTGTACAGTCTGTTTTATAGACTGGATGTGGTCCAGATCAATGAAAACCAGGGGAATCGATCCAACAATTCTAACAAGGAGTACCGGCTGATCAATTGCAACACTAGCGCCATTACCCAGGCTTGTCCCAAAGTGTCCTTTGAACCTATCCCAATTCATTATTGCGCCCCTGCTGGCTTCGCCATCCTGAAGTGTAAAGATAAGAAGTTCAACGGCACCGGGCCCTGCCCTTCTGTGAGTACAGTCCAGTGTACTCACGGGATTAAGCCTGTGGTCAGTACACAGCTGCTGCTGAATGGATCACTGGCTGAGGAAGAAGTGATGATCCGATCCGAGAACATTACTAACAATGCAAAGAATATCCTGGTGCAGTTCAACACCCCTGTCCAGATTAATTGCACTCGCCCAAACAATAACACCCGGAAAAGCATCAGAATTGGACCAGGCCAGGCATTTTACGCCTTCGGGGACATCATTGGAGATATCAGAATGGCACACTGTAATGTGTCCAAGGCCACCTGGAACGAAACACTGGGAAAGGTGGTCAAACAGCTGAGAAAACATTTCGGCAATAACACTATCATTAGGTTTGCTAATAGCTCCGGCGGGGACCTGGAAGTGACTACCCACAGCTTCAACTGCGGAGGCGAGTTCTTTTACTGTAACACATCCGGCCTGTTTAATTCTACATGGATCAGTAACACTTCAGTGCAGGGCTCAAATAGCACCGGGAGCAACGATTCCATCGTGCTGCCTTGCCGCATTAAGCAGATCATTAATATGTGGCAGCGAATTGGACAGGCTATGTATGCACCCCCTATCCAGGGCGTGATTAGATGTGTCTCTAATATCACCGGGCTGATTCTGACACGCGACGGGGGATCTACAAACAGTACAACTGAGACTTTCAGGCCAGGCGGGGGAGACATGAGGGATAACTGGCGCAGCGAACTGTACAAGTATAAAGTGGTCAAAAT CGAGCCC

II: Trimers with PGT121-Class Germline-Targeting Mutations

BG505_SOSIP.D664_JS_3mut_mC (SOSIP-3MUT) (SEQ ID NO: 13)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVANNIIDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 14)GCTGAAAACCTGTGGGTCACTGTCTACTACGGCGTGCCTGTCTGGAAGGATGCTGAAACTACTCTGTTCTGTGCCTCTGATGCTAAAGCCTACGAAACCGAGAAGCACAATGTGTGGGCCACACATGCTTGCGTCCCTACTGACCCAAACCCCCAGGAAATCCACCTGGAGAATGTGACAGAGGAATTCAACATGTGGAAAAACAATATGGTGGAGCAGATGCATACTGACATCATTTCCCTGTGGGATCAGTCTCTGAAGCCTTGCGTGAAACTGACACCACTGTGCGTCACTCTGCAGTGTACTAACGTGGCCAACAATATCATCGACGATATGCGGGGCGAACTGAAGAATTGTTCTTTCAACATGACCACAGAGCTGCGGGACAAGAAACAGAAAGTGTACAGTCTGTTTTATAGACTGGATGTGGTCCAGATCAATGAAAACCAGGGGAATCGAAGTAACAATTCAAACAAGGAGTACCGGCTGATCAATTGCAACACAAGTGCAATTACTCAGGCCTGTCCAAAAGTGTCATTTGAACCTATCCCAATTCATTATTGCGCTCCCGCAGGCTTCGCCATCCTGAAGTGTAAAGATAAGAAGTTCAACGGCACCGGGCCATGCCCTTCAGTGAGCACCGTCCAGTGTACACACGGAATTAAGCCAGTGGTCTCCACACAGCTGCTGCTGAATGGCTCTCTGGCTGAGGAAGAAGTGATGATCAGGAGCGAGAACATTACCAACAATGCAAAGAATATCCTGGTGCAGTTCAACACACCCGTCCAGATTAATTGCACCCGCCCTAACAATAACACACGGAAATCTATCAGAATTGGACCCGGCCAGGCATTTTATGCCACCGGCGACATCATTGGGGATATCAGACAGGCACACTGTAATGTGAGCAAGGCCACTTGGAACGAGACCCTGGGGAAGGTGGTCAAACAGCTGAGAAAGCATTTCGGAAACAACACTATCATCAGGTTTGCCAATAGCTCCGGCGGGGACCTGGAAGTGACTACCCACAGCTTCAACTGCGGAGGCGAGTTCTTTTACTGTAACACAAGTGGCCTGTTTAATTCAACCTGGATCAGCAACACATCCGTGCAGGGATCCAATTCTACAGGCTCTAACGATAGTATCACTCTGCCCTGCCGCATTAAGCAGATCATTAATATGTGGCAGCGAATCGGGCAGGCTATGTATGCACCCCCTATCCAGGGAGTGATTAGATGTGTCAGCAATATCACTGGCCTGATTCTGACCCGCGACGGGGGATCAACTAACAGCACAACTGAGACCTTCCGGCCTGGAGGAGGAGACATGAGGGATAACTGGCGCTCCGAACTGTACAAGTATAAAGTGGTCAAGATCGAGCCACTGGGAGTGGCACCAACCAGATGCAAACGAAGAGTGGTCGGAAGGCGACGACGAAGAAGGGCAGTGGGAATTGGAGCTGTCTTCCTGGGCTTTCTGGGGGCCGCTGGATCTACAATGGGGGCAGCCAGTATGACCCTGACAGTCCAGGCTCGAAATCTGCTGTCAGGAATCGTGCAGCAGCAGAGCAACCTGCTGAGAGCCCCTGAAGCTCAGCAGCATCTGCTGAAGCTGACCGTGTGGGGCATCAAGCAGCTGCAGGCTAGGGTGCTGGCAGTCGAGCGCTACCTGCGAGATCAGCAGCTGCTGGGCATCTGGGGGTGTTCCGGAAAGCTGATTTGCTGTACCAATGTGCCATGGAACTCTAGTTGGAGCAATCGCAACCTGTCCGAAATCTGGGACAATATGACTTGGCTGCAGTGGGATAAGGAGATTAGCAACTACACCCAGATCATCTACGGCCTGCTGGAAGAGTCCCAGAATCAGCAGGAGAAGAACGAGCAGGACCTGCTGGCCCTGG ATBG505_SOSIP.D664_JS_5mut_mC (SOSIP-5MUT) (SEQ ID NO: 15)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNYTPNLTNDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 16)GCTGAAAACCTGTGGGTCACTGTCTACTACGGCGTGCCTGTCTGGAAGGATGCTGAAACTACTCTGTTCTGTGCCTCTGATGCTAAAGCCTACGAAACCGAGAAGCACAATGTGTGGGCCACACATGCTTGCGTCCCTACTGACCCAAACCCCCAGGAAATCCACCTGGAGAATGTGACAGAGGAATTCAACATGTGGAAAAACAATATGGTGGAGCAGATGCATACTGACATCATTTCCCTGTGGGATCAGTCTCTGAAGCCTTGCGTGAAACTGACACCACTGTGCGTCACTCTGCAGTGTACTAACTACACCCCCAATCTGACCAACGATATGCGGGGCGAACTGAAGAATTGTTCTTTCAACATGACCACAGAGCTGCGGGACAAGAAACAGAAAGTGTACAGTCTGTTTTATAGACTGGATGTGGTCCAGATCAATGAAAACCAGGGGAATCGAAGTAACAATTCAAACAAGGAGTACCGGCTGATCAATTGCAACACAAGTGCAATTACTCAGGCCTGTCCAAAAGTGTCATTTGAACCTATCCCAATTCATTATTGCGCTCCCGCAGGCTTCGCCATCCTGAAGTGTAAAGATAAGAAGTTCAACGGCACCGGGCCATGCCCTTCAGTGAGCACCGTCCAGTGTACACACGGAATTAAGCCAGTGGTCTCCACACAGCTGCTGCTGAATGGCTCTCTGGCTGAGGAAGAAGTGATGATCAGGAGCGAGAACATTACCAACAATGCAAAGAATATCCTGGTGCAGTTCAACACACCCGTCCAGATTAATTGCACCCGCCCTAACAATAACACACGGAAATCTATCAGAATTGGACCCGGCCAGGCATTTTATGCCACCGGCGACATCATTGGGGATATCAGACAGGCACACTGTAATGTGAGCAAGGCCACTTGGAACGAGACCCTGGGGAAGGTGGTCAAACAGCTGAGAAAGCATTTCGGAAACAACACTATCATCAGGTTTGCCAATAGCTCCGGCGGGGACCTGGAAGTGACTACCCACAGCTTCAACTGCGGAGGCGAGTTCTTTTACTGTAACACAAGTGGCCTGTTTAATTCAACCTGGATCAGCAACACATCCGTGCAGGGATCCAATTCTACAGGCTCTAACGATAGTATCACTCTGCCCTGCCGCATTAAGCAGATCATTAATATGTGGCAGCGAATCGGGCAGGCTATGTATGCACCCCCTATCCAGGGAGTGATTAGATGTGTCAGCAATATCACTGGCCTGATTCTGACCCGCGACGGGGGATCAACTAACAGCACAACTGAGACCTTCCGGCCTGGAGGAGGAGACATGAGGGATAACTGGCGCTCCGAACTGTACAAGTATAAAGTGGTCAAGATCGAGCCACTGGGAGTGGCACCAACCAGATGCAAACGAAGAGTGGTCGGAAGGCGACGACGAAGAAGGGCAGTGGGAATTGGAGCTGTCTTCCTGGGCTTTCTGGGGGCCGCTGGATCTACAATGGGGGCAGCCAGTATGACCCTGACAGTCCAGGCTCGAAATCTGCTGTCAGGAATCGTGCAGCAGCAGAGCAACCTGCTGAGAGCCCCTGAAGCTCAGCAGCATCTGCTGAAGCTGACCGTGTGGGGCATCAAGCAGCTGCAGGCTAGGGTGCTGGCAGTCGAGCGCTACCTGCGAGATCAGCAGCTGCTGGGCATCTGGGGGTGTTCCGGAAAGCTGATTTGCTGTACCAATGTGCCATGGAACTCTAGTTGGAGCAATCGCAACCTGTCCGAAATCTGGGACAATATGACTTGGCTGCAGTGGGATAAGGAGATTAGCAACTACACCCAGATCATCTACGGCCTGCTGGAAGAGTCCCAGAATCAGCAGGAGAAGAACGAGCAGGACCTGCTGGCCCTGG ATBG505_SOSIP.D664_JS_7mut_mC (SOSIP-7MUT) (SEQ ID NO: 17)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNYAPNLINDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 18)GCTGAAAACCTGTGGGTCACTGTCTACTACGGCGTGCCTGTCTGGAAGGATGCTGAAACTACTCTGTTCTGTGCCTCTGATGCTAAAGCCTACGAAACCGAGAAGCACAATGTGTGGGCCACACATGCTTGCGTCCCTACTGACCCAAACCCCCAGGAAATCCACCTGGAGAATGTGACAGAGGAATTCAACATGTGGAAAAACAATATGGTGGAGCAGATGCATACTGACATCATTTCCCTGTGGGATCAGTCTCTGAAGCCTTGCGTGAAACTGACACCACTGTGCGTCACTCTGCAGTGTACTAACTACGCCCCCAATCTGATCAACGATATGCGGGGCGAACTGAAGAATTGTTCTTTCAACATGACCACAGAGCTGCGGGACAAGAAACAGAAAGTGTACAGTCTGTTTTATAGACTGGATGTGGTCCAGATCAATGAAAACCAGGGGAATCGAAGTAACAATTCAAACAAGGAGTACCGGCTGATCAATTGCAACACAAGTGCAATTACTCAGGCCTGTCCAAAAGTGTCATTTGAACCTATCCCAATTCATTATTGCGCTCCCGCAGGCTTCGCCATCCTGAAGTGTAAAGATAAGAAGTTCAACGGCACCGGGCCATGCCCTTCAGTGAGCACCGTCCAGTGTACACACGGAATTAAGCCAGTGGTCTCCACACAGCTGCTGCTGAATGGCTCTCTGGCTGAGGAAGAAGTGATGATCAGGAGCGAGAACATTACCAACAATGCAAAGAATATCCTGGTGCAGTTCAACACACCCGTCCAGATTAATTGCACCCGCCCTAACAATAACACACGGAAATCTATCAGAATTGGACCCGGCCAGGCATTTTATGCCACCGGCGACATCATTGGGGATATCAGACAGGCACACTGTAATGTGAGCAAGGCCACTTGGAACGAGACCCTGGGGAAGGTGGTCAAACAGCTGAGAAAGCATTTCGGAAACAACACTATCATCAGGTTTGCCAATAGCTCCGGCGGGGACCTGGAAGTGACTACCCACAGCTTCAACTGCGGAGGCGAGTTCTTTTACTGTAACACAAGTGGCCTGTTTAATTCAACCTGGATCAGCAACACATCCGTGCAGGGATCCAATTCTACAGGCTCTAACGATAGTATCACTCTGCCCTGCCGCATTAAGCAGATCATTAATATGTGGCAGCGAATCGGGCAGGCTATGTATGCACCCCCTATCCAGGGAGTGATTAGATGTGTCAGCAATATCACTGGCCTGATTCTGACCCGCGACGGGGGATCAACTAACAGCACAACTGAGACCTTCCGGCCTGGAGGAGGAGACATGAGGGATAACTGGCGCTCCGAACTGTACAAGTATAAAGTGGTCAAGATCGAGCCACTGGGAGTGGCACCAACCAGATGCAAACGAAGAGTGGTCGGAAGGCGACGACGAAGAAGGGCAGTGGGAATTGGAGCTGTCTTCCTGGGCTTTCTGGGGGCCGCTGGATCTACAATGGGGGCAGCCAGTATGACCCTGACAGTCCAGGCTCGAAATCTGCTGTCAGGAATCGTGCAGCAGCAGAGCAACCTGCTGAGAGCCCCTGAAGCTCAGCAGCATCTGCTGAAGCTGACCGTGTGGGGCATCAAGCAGCTGCAGGCTAGGGTGCTGGCAGTCGAGCGCTACCTGCGAGATCAGCAGCTGCTGGGCATCTGGGGGTGTTCCGGAAAGCTGATTTGCTGTACCAATGTGCCATGGAACTCTAGTTGGAGCAATCGCAACCTGTCCGAAATCTGGGACAATATGACTTGGCTGCAGTGGGATAAGGAGATTAGCAACTACACCCAGATCATCTACGGCCTGCTGGAAGAGTCCCAGAATCAGCAGGAGAAGAACGAGCAGGACCTGCTGGCCCTGG ATBG505_SOSIP.D664_JS_10mut_mC (SOSIP-10MUT) (SEQ ID NO: 19)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNYAPFLINDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYAFGDIIGDIRMAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 20)GCTGAAAACCTGTGGGTCACTGTCTACTACGGCGTGCCTGTCTGGAAGGATGCTGAAACTACTCTGTTCTGTGCCTCTGATGCTAAAGCCTACGAAACCGAGAAGCACAATGTGTGGGCCACACATGCTTGCGTCCCTACTGACCCAAACCCCCAGGAAATCCACCTGGAGAATGTGACAGAGGAATTCAACATGTGGAAAAACAATATGGTGGAGCAGATGCATACTGACATCATTTCCCTGTGGGATCAGTCTCTGAAGCCTTGCGTGAAACTGACACCACTGTGCGTCACTCTGCAGTGTACTAACTACGCCCCCTTTCTGATCAACGATATGCGGGGCGAACTGAAGAATTGTTCTTTCAACATGACCACAGAGCTGCGGGACAAGAAACAGAAAGTGTACAGTCTGTTTTATAGACTGGATGTGGTCCAGATCAATGAAAACCAGGGGAATCGAAGTAACAATTCAAACAAGGAGTACCGGCTGATCAATTGCAACACAAGTGCAATTACTCAGGCCTGTCCAAAAGTGTCATTTGAACCTATCCCAATTCATTATTGCGCTCCCGCAGGCTTCGCCATCCTGAAGTGTAAAGATAAGAAGTTCAACGGCACCGGGCCATGCCCTTCAGTGAGCACCGTCCAGTGTACACACGGAATTAAGCCAGTGGTCTCCACACAGCTGCTGCTGAATGGCTCTCTGGCTGAGGAAGAAGTGATGATCAGGAGCGAGAACATTACCAACAATGCAAAGAATATCCTGGTGCAGTTCAACACACCCGTCCAGATTAATTGCACCCGCCCTAACAATAACACACGGAAATCTATCAGAATTGGACCCGGCCAGGCATTTTATGCCTTTGGCGACATCATTGGGGATATCAGAATGGCACACTGTAATGTGAGCAAGGCCACTTGGAACGAGACCCTGGGGAAGGTGGTCAAACAGCTGAGAAAGCATTTCGGAAACAACACTATCATCAGGTTTGCCAATAGCTCCGGCGGGGACCTGGAAGTGACTACCCACAGCTTCAACTGCGGAGGCGAGTTCTTTTACTGTAACACAAGTGGCCTGTTTAATTCAACCTGGATCAGCAACACATCCGTGCAGGGATCCAATTCTACAGGCTCTAACGATAGTATCACTCTGCCCTGCCGCATTAAGCAGATCATTAATATGTGGCAGCGAATCGGGCAGGCTATGTATGCACCCCCTATCCAGGGAGTGATTAGATGTGTCAGCAATATCACTGGCCTGATTCTGACCCGCGACGGGGGATCAACTAACAGCACAACTGAGACCTTCCGGCCTGGAGGAGGAGACATGAGGGATAACTGGCGCTCCGAACTGTACAAGTATAAAGTGGTCAAGATCGAGCCACTGGGAGTGGCACCAACCAGATGCAAACGAAGAGTGGTCGGAAGGCGACGACGAAGAAGGGCAGTGGGAATTGGAGCTGTCTTCCTGGGCTTTCTGGGGGCCGCTGGATCTACAATGGGGGCAGCCAGTATGACCCTGACAGTCCAGGCTCGAAATCTGCTGTCAGGAATCGTGCAGCAGCAGAGCAACCTGCTGAGAGCCCCTGAAGCTCAGCAGCATCTGCTGAAGCTGACCGTGTGGGGCATCAAGCAGCTGCAGGCTAGGGTGCTGGCAGTCGAGCGCTACCTGCGAGATCAGCAGCTGCTGGGCATCTGGGGGTGTTCCGGAAAGCTGATTTGCTGTACCAATGTGCCATGGAACTCTAGTTGGAGCAATCGCAACCTGTCCGAAATCTGGGACAATATGACTTGGCTGCAGTGGGATAAGGAGATTAGCAACTACACCCAGATCATCTACGGCCTGCTGGAAGAGTCCCAGAATCAGCAGGAGAAGAACGAGCAGGACCTGCTGGCCCTGG ATBG505_SOSIP.D664_JS_10mut2A_mC (SEQ ID NO: 21)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNYAPFLINNMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYAFGDIIGDIRMAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 22)GCTGAAAACCTGTGGGTCACTGTCTACTACGGCGTGCCTGTCTGGAAGGATGCTGAAACTACTCTGTTCTGTGCCTCTGATGCTAAAGCCTACGAAACCGAGAAGCACAATGTGTGGGCCACACATGCTTGCGTCCCTACTGACCCAAACCCCCAGGAAATCCACCTGGAGAATGTGACAGAGGAATTCAACATGTGGAAAAACAATATGGTGGAGCAGATGCATACTGACATCATTTCCCTGTGGGATCAGTCTCTGAAGCCTTGCGTGAAACTGACACCACTGTGCGTCACTCTGCAGTGTACTAACTACGCCCCCTTTCTGATCAACAACATGCGGGGCGAACTGAAGAATTGTTCTTTCAACATGACCACAGAGCTGCGGGACAAGAAACAGAAAGTGTACAGTCTGTTTTATAGACTGGATGTGGTCCAGATCAATGAAAACCAGGGGAATCGAAGTAACAATTCAAACAAGGAGTACCGGCTGATCAATTGCAACACAAGTGCAATTACTCAGGCCTGTCCAAAAGTGTCATTTGAACCTATCCCAATTCATTATTGCGCTCCCGCAGGCTTCGCCATCCTGAAGTGTAAAGATAAGAAGTTCAACGGCACCGGGCCATGCCCTTCAGTGAGCACCGTCCAGTGTACACACGGAATTAAGCCAGTGGTCTCCACACAGCTGCTGCTGAATGGCTCTCTGGCTGAGGAAGAAGTGATGATCAGGAGCGAGAACATTACCAACAATGCAAAGAATATCCTGGTGCAGTTCAACACACCCGTCCAGATTAATTGCACCCGCCCTAACAATAACACACGGAAATCTATCAGAATTGGACCCGGCCAGGCATTTTATGCCTTTGGCGACATCATTGGGGATATCAGAATGGCACACTGTAATGTGAGCAAGGCCACTTGGAACGAGACCCTGGGGAAGGTGGTCAAACAGCTGAGAAAGCATTTCGGAAACAACACTATCATCAGGTTTGCCAATAGCTCCGGCGGGGACCTGGAAGTGACTACCCACAGCTTCAACTGCGGAGGCGAGTTCTTTTACTGTAACACAAGTGGCCTGTTTAATTCAACCTGGATCAGCAACACATCCGTGCAGGGATCCAATTCTACAGGCTCTAACGATAGTATCACTCTGCCCTGCCGCATTAAGCAGATCATTAATATGTGGCAGCGAATCGGGCAGGCTATGTATGCACCCCCTATCCAGGGAGTGATTAGATGTGTCAGCAATATCACTGGCCTGATTCTGACCCGCGACGGGGGATCAACTAACAGCACAACTGAGACCTTCCGGCCTGGAGGAGGAGACATGAGGGATAACTGGCGCTCCGAACTGTACAAGTATAAAGTGGTCAAGATCGAGCCACTGGGAGTGGCACCAACCAGATGCAAACGAAGAGTGGTCGGAAGGCGACGACGAAGAAGGGCAGTGGGAATTGGAGCTGTCTTCCTGGGCTTTCTGGGGGCCGCTGGATCTACAATGGGGGCAGCCAGTATGACCCTGACAGTCCAGGCTCGAAATCTGCTGTCAGGAATCGTGCAGCAGCAGAGCAACCTGCTGAGAGCCCCTGAAGCTCAGCAGCATCTGCTGAAGCTGACCGTGTGGGGCATCAAGCAGCTGCAGGCTAGGGTGCTGGCAGTCGAGCGCTACCTGCGAGATCAGCAGCTGCTGGGCATCTGGGGGTGTTCCGGAAAGCTGATTTGCTGTACCAATGTGCCATGGAACTCTAGTTGGAGCAATCGCAACCTGTCCGAAATCTGGGACAATATGACTTGGCTGCAGTGGGATAAGGAGATTAGCAACTACACCCAGATCATCTACGGCCTGCTGGAAGAGTCCCAGAATCAGCAGGAGAAGAACGAGCAGGACCTGCTGGCCCTGG ATBG505_SOSIP.D664_JS_11mut2A_mC (SEQ ID NO: 23)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNYAPNLLSNMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYAFGDIIGDIRMAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSIVLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 24)GCTGAAAACCTGTGGGTCACTGTCTACTACGGCGTGCCTGTCTGGAAGGATGCTGAAACTACTCTGTTCTGTGCCTCTGATGCTAAAGCCTACGAAACCGAGAAGCACAATGTGTGGGCCACACATGCTTGCGTCCCTACTGACCCAAACCCCCAGGAAATCCACCTGGAGAATGTGACAGAGGAATTCAACATGTGGAAAAACAATATGGTGGAGCAGATGCATACTGACATCATTTCCCTGTGGGATCAGTCTCTGAAGCCTTGCGTGAAACTGACACCACTGTGCGTCACTCTGCAGTGTACTAACTACGCCCCAAACCTGCTGTCCAATATGCGGGGCGAACTGAAGAATTGTTCTTTCAACATGACCACAGAGCTGCGGGACAAGAAACAGAAAGTGTACAGTCTGTTTTATAGACTGGATGTGGTCCAGATCAATGAAAACCAGGGGAATCGAAGTAACAATTCAAACAAGGAGTACCGGCTGATCAATTGCAACACAAGTGCAATTACTCAGGCCTGTCCAAAAGTGTCATTTGAACCTATCCCAATTCATTATTGCGCTCCCGCAGGCTTCGCCATCCTGAAGTGTAAAGATAAGAAGTTCAACGGCACCGGGCCATGCCCTTCAGTGAGCACCGTCCAGTGTACACACGGAATTAAGCCAGTGGTCTCCACACAGCTGCTGCTGAATGGCTCTCTGGCTGAGGAAGAAGTGATGATCAGGAGCGAGAACATTACCAACAATGCAAAGAATATCCTGGTGCAGTTCAACACACCCGTCCAGATTAATTGCACCCGCCCTAACAATAACACACGGAAATCTATCAGAATTGGACCCGGCCAGGCATTTTATGCCTTTGGCGACATCATTGGGGATATCAGAATGGCACACTGTAATGTGAGCAAGGCCACTTGGAACGAGACCCTGGGGAAGGTGGTCAAACAGCTGAGAAAGCATTTCGGAAACAACACTATCATCAGGTTTGCCAATAGCTCCGGCGGGGACCTGGAAGTGACTACCCACAGCTTCAACTGCGGAGGCGAGTTCTTTTACTGTAACACAAGTGGCCTGTTTAATTCAACCTGGATCAGCAACACATCCGTGCAGGGATCCAATTCTACAGGCTCTAACGATAGTATCGTGCTGCCCTGCCGCATTAAGCAGATCATTAATATGTGGCAGCGAATCGGGCAGGCTATGTATGCACCCCCTATCCAGGGAGTGATTAGATGTGTCAGCAATATCACTGGCCTGATTCTGACCCGCGACGGGGGATCAACTAACAGCACAACTGAGACCTTCCGGCCTGGAGGAGGAGACATGAGGGATAACTGGCGCTCCGAACTGTACAAGTATAAAGTGGTCAAGATCGAGCCACTGGGAGTGGCACCAACCAGATGCAAACGAAGAGTGGTCGGAAGGCGACGACGAAGAAGGGCAGTGGGAATTGGAGCTGTCTTCCTGGGCTTTCTGGGGGCCGCTGGATCTACAATGGGGGCAGCCAGTATGACCCTGACAGTCCAGGCTCGAAATCTGCTGTCAGGAATCGTGCAGCAGCAGAGCAACCTGCTGAGAGCCCCTGAAGCTCAGCAGCATCTGCTGAAGCTGACCGTGTGGGGCATCAAGCAGCTGCAGGCTAGGGTGCTGGCAGTCGAGCGCTACCTGCGAGATCAGCAGCTGCTGGGCATCTGGGGGTGTTCCGGAAAGCTGATTTGCTGTACCAATGTGCCATGGAACTCTAGTTGGAGCAATCGCAACCTGTCCGAAATCTGGGACAATATGACTTGGCTGCAGTGGGATAAGGAGATTAGCAACTACACCCAGATCATCTACGGCCTGCTGGAAGAGTCCCAGAATCAGCAGGAGAAGAACGAGCAGGACCTGCTGGCCCTGG AT

III: Stabilized Trimers with Improved Expression, Thermal Stability,and/or Antigenic Profile for Mature bnAbs

BG505_SOSIP_D664_MD39_mC (BG505 SOSIP-MD39 or MD39) (SEQ ID NO: 25)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLAL D (SEQID NO: 26) GCCGAAAATCTGTGGGTGACTGTCTACTATGGCGTGCCTGTCTGGAAGGACGCCGAGACCACACTGTTCTGTGCTTCCGATGCTAAGGCATACGAAACCGAGAAACACAACGTGTGGGCAACCCATGCCTGCGTCCCAACAGACCCAAACCCCCAGGAAATCCACCTGGAGAATGTGACCGAGGAGTTCAACATGTGGAAGAACAATATGGTGGAACAGATGCATGAGGACATCATTTCCCTGTGGGATCAGTCTCTGAAGCCTTGCGTGAAACTGACCCCACTGTGCGTGACACTGCAGTGTACAAACGTCACTAACAATATCACCGACGATATGCGGGGCGAACTGAAGAATTGTTCTTTCAACATGACTACCGAGCTGAGGGACAAGAAACAGAAAGTGTACAGTCTGTTTTATCGCCTGGATGTGGTCCAGATCAATGAAAACCAGGGGAATCGAAGTAACAATTCAAACAAGGAGTACCGGCTGATTAATTGCAACACCAGTGCCATCACACAGGCTTGTCCAAAAGTGTCATTCGAGCCTATCCCAATTCATTATTGCGCCCCCGCTGGCTTCGCCATCCTGAAGTGTAAAGATAAGAAGTTCAACGGCACCGGGCCATGCCCTTCAGTGAGCACTGTCCAGTGTACCCACGGAATTAAGCCTGTGGTCTCCACACAGCTGCTGCTGAATGGCTCTCTGGCTGAGGAAGAAGTGATCATTAGGTCTGAGAACATCACTAACAATGCAAAGAATATTCTGGTGCAGCTGAACACCCCCGTCCAGATCAATTGCACTCGCCCTAACAATAACACCGTGAAATCTATCCGAATTGGACCCGGCCAGGCTTTTTATTACACCGGCGACATCATTGGCGACATCAGACAGGCACACTGCAATGTGAGCAAGGCCACATGGAACGAGACTCTGGGGAAGGTGGTCAAACAGCTGCGCAAACATTTCGGAAATAACACAATCATTCGATTTGCACAGAGCAGCGGAGGGGACCTGGAAGTGACAACTCACAGCTTCAATTGCGGAGGCGAGTTCTTTTACTGTAACACTAGTGGCCTGTTTAATTCAACTTGGATCAGCAACACCTCCGTGCAGGGCAGCAACAGCACCGGCTCTAACGATAGTATCACACTGCCATGTCGGATTAAGCAGATCATTAACATGTGGCAGAGAATCGGGCAGGCCATGTATGCACCCCCTATCCAGGGAGTGATTCGATGCGTGAGCAATATCACAGGCCTGATTCTGACTAGAGACGGGGGATCAACAAACAGCACCACAGAGACTTTCCGGCCCGGCGGAGGAGACATGCGAGATAACTGGAGATCCGAACTGTACAAGTATAAAGTGGTCAAGATCGAGCCACTGGGCGTGGCTCCCACCAGATGCAAACGAAGAGTGGTCGGGAGGCGCCGACGGAGAAGGGCTGTGGGGATTGGAGCAGTCAGCCTGGGCTTTCTGGGGGCCGCTGGATCTACAATGGGGGCAGCCAGTATGACTCTGACCGTGCAGGCCAGGAATCTGCTGTCAGGAATCGTGCAGCAGCAGAGCAACCTGCTGAGGGCTCCCGAACCCCAGCAGCACCTGCTGAAGGACACCCACTGGGGCATCAAGCAGCTGCAGGCAAGAGTGCTGGCCGTCGAGCATTACCTGAGGGATCAGCAGCTGCTGGGCATCTGGGGATGCAGCGGAAAGCTGATTTGCTGTACAAATGTGCCTTGGAACTCTAGTTGGAGCAATCGCAACCTGTCCGAAATCTGGGACAATATGACATGGCTGCAGTGGGATAAGGAGATTAGCAACTACACTCAGATCATCTACGGCCTGCTGGAAGAGTCCCAGAATCAGCAGGAGAAGAACGAGCAGGACCTGCTGGCCCTGGATBG505_SOSIP_D664_MD16_mC stabilizes the V3 loop (SEQ ID NO: 27)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGWAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKDTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLAL D (SEQID NO: 28) GCTGAAAACCTGTGGGTCACTGTCTACTACGGCGTGCCTGTCTGGAAGGATGCTGAAACTACTCTGTTCTGTGCCTCTGATGCTAAAGCCTACGAAACCGAGAAGCACAATGTGTGGGCCACACATGCTTGCGTCCCTACTGACCCAAACCCCCAGGAAATCCACCTGGAGAATGTGACAGAGGAATTCAACATGTGGAAAAACAATATGGTGGAGCAGATGCATACTGACATCATTTCCCTGTGGGATCAGTCTCTGAAGCCTTGCGTGAAACTGACACCACTGTGCGTCACTCTGCAGTGTACTAACGTGACCAACAATATCACCGACGATATGCGGGGCGAACTGAAGAATTGTTCTTTCAACATGACCACAGAGCTGCGGGACAAGAAACAGAAAGTGTACAGTCTGTTTTATAGACTGGATGTGGTCCAGATCAATGAAAACCAGGGGAATCGAAGTAACAATTCAAACAAGGAGTACCGGCTGATCAATTGCAACACAAGTGCAATTACTCAGGCCTGTCCAAAAGTGTCATTTGAACCTATCCCAATTCATTATTGCGCTCCCGCAGGCTGGGCCATCCTGAAGTGTAAAGATAAGAAGTTCAACGGCACCGGGCCATGCCCTTCAGTGAGCACCGTCCAGTGTACACACGGAATTAAGCCAGTGGTCTCCACACAGCTGCTGCTGAATGGCTCTCTGGCTGAGGAAGAAGTGATGATCAGGAGCGAGAACATTACCAACAATGCAAAGAATATCCTGGTGCAGTTCAACACACCCGTCCAGATTAATTGCACCCGCCCTAACAATAACACAGTGAAATCTATCAGAATTGGACCCGGCCAGGCATTTTATTACACCGGCGACATCATTGGGGATATCAGACAGGCACACTGTAATGTGAGCAAGGCCACTTGGAACGAGACCCTGGGGAAGGTGGTCAAACAGCTGAGAAAGCATTTCGGAAACAACACTATCATCAGGTTTGCCAATAGCTCCGGCGGGGACCTGGAAGTGACTACCCACAGCTTCAACTGCGGAGGCGAGTTCTTTTACTGTAACACAAGTGGCCTGTTTAATTCAACCTGGATCAGCAACACATCCGTGCAGGGATCCAATTCTACAGGCTCTAACGATAGTATCACTCTGCCCTGCCGCATTAAGCAGATCATTAATATGTGGCAGCGAATCGGGCAGGCTATGTATGCACCCCCTATCCAGGGAGTGATTAGATGTGTCAGCAATATCACTGGCCTGATTCTGACCCGCGACGGGGGATCAACTAACAGCACAACTGAGACCTTCCGGCCTGGAGGAGGAGACATGAGGGATAACTGGCGCTCCGAACTGTACAAGTATAAAGTGGTCAAGATCGAGCCACTGGGAGTGGCACCAACCAGATGCAAACGAAGAGTGGTCGGAAGGCGACGACGAAGAAGGGCAGTGGGAATTGGAGCTGTCTTCCTGGGCTTTCTGGGGGCCGCTGGATCTACAATGGGGGCAGCCAGTATGACCCTGACAGTCCAGGCTCGAAATCTGCTGTCAGGAATCGTGCAGCAGCAGAGCAACCTGCTGAGAGCCCCTGAAGCTCAGCAGCATCTGCTGAAGGACACCGTGTGGGGCATCAAGCAGCTGCAGGCTAGGGTGCTGGCAGTCGAGCGCTACCTGCGAGATCAGCAGCTGCTGGGCATCTGGGGGTGTTCCGGAAAGCTGATTTGCTGTACCAATGTGCCATGGAACTCTAGTTGGAGCAATCGCAACCTGTCCGAAATCTGGGACAATATGACTTGGCTGCAGTGGGATAAGGAGATTAGCAACTACACCCAGATCATCTACGGCCTGCTGGAAGAGTCCCAGAATCAGCAGGAGAAGAACGAGCAGGACCTGCTGGCCCTGGATBG505_SOSIP_D664_split1_1_mC increases melting temp 10 C. (SEQ ID NO:29) AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHECVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIIELWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLAL D (SEQID NO: 30) GCTGAAAACCTGTGGGTCACTGTCTACTACGGCGTGCCTGTCTGGAAGGATGCTGAAACTACTCTGTTCTGTGCCTCTGATGCTAAAGCCTACGAAACCGAGAAGCACAATGTGTGGGCCACACATGAGTGCGTCCCTACTGACCCAAACCCCCAGGAAATCCACCTGGAGAATGTGACAGAGGAATTCAACATGTGGAAAAACAATATGGTGGAGCAGATGCATACTGACATCATTGAGCTGTGGGATCAGTCTCTGAAGCCTTGCGTGAAACTGACACCACTGTGCGTCACTCTGCAGTGTACTAACGTGACCAACAATATCACCGACGATATGCGGGGCGAACTGAAGAATTGTTCTTTCAACATGACCACAGAGCTGCGGGACAAGAAACAGAAAGTGTACAGTCTGTTTTATAGACTGGATGTGGTCCAGATCAATGAAAACCAGGGGAATCGAAGTAACAATTCAAACAAGGAGTACCGGCTGATCAATTGCAACACAAGTGCAATTACTCAGGCCTGTCCAAAAGTGTCATTTGAACCTATCCCAATTCATTATTGCGCTCCCGCAGGCTTCGCCATCCTGAAGTGTAAAGATAAGAAGTTCAACGGCACCGGGCCATGCCCTTCAGTGAGCACCGTCCAGTGTACACACGGAATTAAGCCAGTGGTCTCCACACAGCTGCTGCTGAATGGCTCTCTGGCTGAGGAAGAAGTGATGATCAGGAGCGAGAACATTACCAACAATGCAAAGAATATCCTGGTGCAGTTCAACACACCCGTCCAGATTAATTGCACCCGCCCTAACAATAACACACGGAAATCTATCAGAATTGGACCCGGCCAGGCATTTTATGCCACCGGCGACATCATTGGGGATATCAGACAGGCACACTGTAATGTGAGCAAGGCCACTTGGAACGAGACCCTGGGGAAGGTGGTCAAACAGCTGAGAAAGCATTTCGGAAACAACACTATCATCAGGTTTGCCAATAGCTCCGGCGGGGACCTGGAAGTGACTACCCACAGCTTCAACTGCGGAGGCGAGTTCTTTTACTGTAACACAAGTGGCCTGTTTAATTCAACCTGGATCAGCAACACATCCGTGCAGGGATCCAATTCTACAGGCTCTAACGATAGTATCACTCTGCCCTGCCGCATTAAGCAGATCATTAATATGTGGCAGCGAATCGGGCAGGCTATGTATGCACCCCCTATCCAGGGAGTGATTAGATGTGTCAGCAATATCACTGGCCTGATTCTGACCCGCGACGGGGGATCAACTAACAGCACAACTGAGACCTTCCGGCCTGGAGGAGGAGACATGAGGGATAACTGGCGCTCCGAACTGTACAAGTATAAAGTGGTCAAGATCGAGCCACTGGGAGTGGCACCAACCAGATGCAAACGAAGAGTGGTCGGAAGGCGACGACGAAGAAGGGCAGTGGGAATTGGAGCTGTCTTCCTGGGCTTTCTGGGGGCCGCTGGATCTACAATGGGGGCAGCCAGTATGACCCTGACAGTCCAGGCTCGAAATCTGCTGTCAGGAATCGTGCAGCAGCAGAGCAACCTGCTGAGAGCCCCTGAAGCTCAGCAGCATCTGCTGAAGCTGACCGTGTGGGGCATCAAGCAGCTGCAGGCTAGGGTGCTGGCAGTCGAGCGCTACCTGCGAGATCAGCAGCTGCTGGGCATCTGGGGGTGTTCCGGAAAGCTGATTTGCTGTACCAATGTGCCATGGAACTCTAGTTGGAGCAATCGCAACCTGTCCGAAATCTGGGACAATATGACTTGGCTGCAGTGGGATAAGGAGATTAGCAACTACACCCAGATCATCTACGGCCTGCTGGAAGAGTCCCAGAATCAGCAGGAGAAGAACGAGCAGGACCTGCTGGCCCTGGATBG505_SOSIP_D664_MD39C_mC highest melting temp 82.5 C. (SEQ ID NO: 31)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHECVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIIELWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKLTVWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLAL D (SEQID NO: 32) GCCGAGAATCTGTGGGTGACCGTGTACTATGGCGTGCCCGTGTGGAAGGACGCAGAGACCACACTGTTCTGCGCCAGCGATGCCAAGGCCTACGAGACAGAGAAGCACAACGTGTGGGCCACCCACGAGTGCGTGCCTACAGACCCAAACCCCCAGGAGATCCACCTGGAGAATGTGACCGAGGAGTTTAACATGTGGAAGAACAATATGGTGGAGCAGATGCACACAGACATCATCGAGCTGTGGGATCAGTCCCTGAAGCCCTGCGTGAAGCTGACCCCTCTGTGCGTGACACTGCAGTGTACCAACGTGACAAACAATATCACCGACGATATGAGGGGCGAGCTGAAGAATTGTAGCTTCAACATGACCACAGAGCTGCGGGACAAGAAGCAGAAGGTGTACTCCCTGTTTTATAGACTGGATGTGGTGCAGATCAATGAGAACCAGGGCAATAGGTCTAACAATAGCAACAAGGAGTACCGCCTGATCAATTGCAACACCTCTGCCATCACACAGGCCTGTCCTAAGGTGAGCTTCGAGCCTATCCCAATCCACTATTGCGCCCCAGCCGGCTTCGCCATCCTGAAGTGTAAGGATAAGAAGTTTAACGGCACCGGCCCATGCCCTTCCGTGTCTACCGTGCAGTGTACACACGGCATCAAGCCTGTGGTGAGCACACAGCTGCTGCTGAATGGCTCCCTGGCCGAGGAGGAAGTGATCATCCGGAGCGAGAACATCACCAACAATGCCAAGAATATCCTGGTGCAGCTGAACACACCAGTGCAGATCAATTGCACCAGGCCCAACAATAACACAGTGAAGTCTATCCGCATCGGCCCAGGCCAGGCCTTTTACTATACCGGCGACATCATCGGCGACATCAGACAGGCCCACTGTAATGTGAGCAAGGCCACCTGGAACGAGACACTGGGCAAGGTGGTGAAGCAGCTGCGGAAGCACTTCGGCAATAACACCATCATCAGATTTGCACAGAGCAGCGGAGGCGACCTGGAGGTGACCACACACTCCTTCAATTGCGGCGGCGAGTTCTTTTACTGTAACACATCCGGCCTGTTTAATTCTACCTGGATCTCCAACACATCTGTGCAGGGCAGCAATTCCACCGGCAGCAACGATTCCATCACACTGCCATGCAGGATCAAGCAGATCATCAACATGTGGCAGAGGATCGGACAGGCAATGTATGCACCACCTATCCAGGGCGTGATCAGATGCGTGAGCAATATCACCGGCCTGATCCTGACACGCGACGGAGGCTCTACCAACAGCACCACAGAGACATTCAGGCCCGGCGGAGGCGACATGAGGGATAACTGGAGATCTGAGCTGTACAAGTATAAGGTGGTGAAGATCGAGCCTCTGGGAGTGGCACCAACCAGGTGCAAGAGGAGAGTGGTGGGCAGGCGCCGGAGAAGGCGCGCAGTGGGCATCGGAGCCGTGAGCCTGGGCTTTCTGGGAGCAGCAGGCAGCACAATGGGCGCAGCCAGCATGACCCTGACAGTGCAGGCCCGGAATCTGCTGTCCGGCATCGTGCAGCAGCAGTCTAACCTGCTGAGAGCCCCAGAGCCCCAGCAGCACCTGCTGAAGCTGACCGTGTGGGGCATCAAGCAGCTGCAGGCCAGAGTGCTGGCCGTGGAGCACTACCTGAGAGATCAGCAGCTGCTGGGCATCTGGGGATGCTCCGGCAAGCTGATCTGCTGTACCAATGTGCCCTGGAACTCTAGCTGGAGCAATAGAAACCTGTCCGAGATCTGGGACAATATGACCTGGCTGCAGTGGGATAAGGAGATCTCCAACTACACACAGATCATCTATGGCCTGCTGGAGGAGTCTCAGAATCAGCAGGAGAAGAACGAGCAGGACCTGCTGGCCCTGGATBG505_SOSIP_D664_MD9_mC improved yield/stability (SEQ ID NO: 33)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPERQQHLLKDTVWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLAL D (SEQID NO: 34) GCTGAAAACCTGTGGGTCACTGTCTACTACGGCGTGCCTGTCTGGAAGGATGCTGAAACTACTCTGTTCTGTGCCTCTGATGCTAAAGCCTACGAAACCGAGAAGCACAATGTGTGGGCCACACATGCTTGCGTCCCTACTGACCCAAACCCCCAGGAAATCCACCTGGAGAATGTGACAGAGGAATTCAACATGTGGAAAAACAATATGGTGGAGCAGATGCATACTGACATCATTTCCCTGTGGGATCAGTCTCTGAAGCCTTGCGTGAAACTGACACCACTGTGCGTCACTCTGCAGTGTACTAACGTGACCAACAATATCACCGACGATATGCGGGGCGAACTGAAGAATTGTTCTTTCAACATGACCACAGAGCTGCGGGACAAGAAACAGAAAGTGTACAGTCTGTTTTATAGACTGGATGTGGTCCAGATCAATGAAAACCAGGGGAATCGAAGTAACAATTCAAACAAGGAGTACCGGCTGATCAATTGCAACACAAGTGCAATTACTCAGGCCTGTCCAAAAGTGTCATTTGAACCTATCCCAATTCATTATTGCGCTCCCGCAGGCTTCGCCATCCTGAAGTGTAAAGATAAGAAGTTCAACGGCACCGGGCCATGCCCTTCAGTGAGCACCGTCCAGTGTACACACGGAATTAAGCCAGTGGTCTCCACACAGCTGCTGCTGAATGGCTCTCTGGCTGAGGAAGAAGTGATGATCAGGAGCGAGAACATTACCAACAATGCAAAGAATATCCTGGTGCAGTTCAACACACCCGTCCAGATTAATTGCACCCGCCCTAACAATAACACACGGAAATCTATCAGAATTGGACCCGGCCAGGCATTTTATGCCACCGGCGACATCATTGGGGATATCAGACAGGCACACTGTAATGTGAGCAAGGCCACTTGGAACGAGACCCTGGGGAAGGTGGTCAAACAGCTGAGAAAGCATTTCGGAAACAACACTATCATCAGGTTTGCCAATAGCTCCGGCGGGGACCTGGAAGTGACTACCCACAGCTTCAACTGCGGAGGCGAGTTCTTTTACTGTAACACAAGTGGCCTGTTTAATTCAACCTGGATCAGCAACACATCCGTGCAGGGATCCAATTCTACAGGCTCTAACGATAGTATCACTCTGCCCTGCCGCATTAAGCAGATCATTAATATGTGGCAGCGAATCGGGCAGGCTATGTATGCACCCCCTATCCAGGGAGTGATTAGATGTGTCAGCAATATCACTGGCCTGATTCTGACCCGCGACGGGGGATCAACTAACAGCACAACTGAGACCTTCCGGCCTGGAGGAGGAGACATGAGGGATAACTGGCGCTCCGAACTGTACAAGTATAAAGTGGTCAAGATCGAGCCACTGGGAGTGGCACCAACCAGATGCAAACGAAGAGTGGTCGGAAGGCGACGACGAAGAAGGGCAGTGGGAATTGGAGCTGTCTTCCTGGGCTTTCTGGGGGCCGCTGGATCTACAATGGGGGCAGCCAGTATGACCCTGACAGTCCAGGCTCGAAATCTGCTGTCAGGAATCGTGCAGCAGCAGAGCAACCTGCTGAGAGCCCCTGAACGGCAGCAGCATCTGCTGAAGGACACCGTGTGGGGCATCAAGCAGCTGCAGGCTAGGGTGCTGGCAGTCGAGCACTACCTGCGAGATCAGCAGCTGCTGGGCATCTGGGGGTGTTCCGGAAAGCTGATTTGCTGTACCAATGTGCCATGGAACTCTAGTTGGAGCAATCGCAACCTGTCCGAAATCTGGGACAATATGACTTGGCTGCAGTGGGATAAGGAGATTAGCAACTACACCCAGATCATCTACGGCCTGCTGGAAGAGTCCCAGAATCAGCAGGAGAAGAACGAGCAGGACCTGCTGGCCCTGGATBG505_SOSIP_D664_MD2_mC improved yield (SEQ ID NO: 35)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKDTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLAL D (SEQID NO: 36) GCTGAAAACCTGTGGGTCACTGTCTACTACGGCGTGCCTGTCTGGAAGGATGCTGAAACTACTCTGTTCTGTGCCTCTGATGCTAAAGCCTACGAAACCGAGAAGCACAATGTGTGGGCCACACATGCTTGCGTCCCTACTGACCCAAACCCCCAGGAAATCCACCTGGAGAATGTGACAGAGGAATTCAACATGTGGAAAAACAATATGGTGGAGCAGATGCATACTGACATCATTTCCCTGTGGGATCAGTCTCTGAAGCCTTGCGTGAAACTGACACCACTGTGCGTCACTCTGCAGTGTACTAACGTGACCAACAATATCACCGACGATATGCGGGGCGAACTGAAGAATTGTTCTTTCAACATGACCACAGAGCTGCGGGACAAGAAACAGAAAGTGTACAGTCTGTTTTATAGACTGGATGTGGTCCAGATCAATGAAAACCAGGGGAATCGAAGTAACAATTCAAACAAGGAGTACCGGCTGATCAATTGCAACACAAGTGCAATTACTCAGGCCTGTCCAAAAGTGTCATTTGAACCTATCCCAATTCATTATTGCGCTCCCGCAGGCTTCGCCATCCTGAAGTGTAAAGATAAGAAGTTCAACGGCACCGGGCCATGCCCTTCAGTGAGCACCGTCCAGTGTACACACGGAATTAAGCCAGTGGTCTCCACACAGCTGCTGCTGAATGGCTCTCTGGCTGAGGAAGAAGTGATGATCAGGAGCGAGAACATTACCAACAATGCAAAGAATATCCTGGTGCAGTTCAACACACCCGTCCAGATTAATTGCACCCGCCCTAACAATAACACACGGAAATCTATCAGAATTGGACCCGGCCAGGCATTTTATGCCACCGGCGACATCATTGGGGATATCAGACAGGCACACTGTAATGTGAGCAAGGCCACTTGGAACGAGACCCTGGGGAAGGTGGTCAAACAGCTGAGAAAGCATTTCGGAAACAACACTATCATCAGGTTTGCCAATAGCTCCGGCGGGGACCTGGAAGTGACTACCCACAGCTTCAACTGCGGAGGCGAGTTCTTTTACTGTAACACAAGTGGCCTGTTTAATTCAACCTGGATCAGCAACACATCCGTGCAGGGATCCAATTCTACAGGCTCTAACGATAGTATCACTCTGCCCTGCCGCATTAAGCAGATCATTAATATGTGGCAGCGAATCGGGCAGGCTATGTATGCACCCCCTATCCAGGGAGTGATTAGATGTGTCAGCAATATCACTGGCCTGATTCTGACCCGCGACGGGGGATCAACTAACAGCACAACTGAGACCTTCCGGCCTGGAGGAGGAGACATGAGGGATAACTGGCGCTCCGAACTGTACAAGTATAAAGTGGTCAAGATCGAGCCACTGGGAGTGGCACCAACCAGATGCAAACGAAGAGTGGTCGGAAGGCGACGACGAAGAAGGGCAGTGGGAATTGGAGCTGTCTTCCTGGGCTTTCTGGGGGCCGCTGGATCTACAATGGGGGCAGCCAGTATGACCCTGACAGTCCAGGCTCGAAATCTGCTGTCAGGAATCGTGCAGCAGCAGAGCAACCTGCTGAGAGCCCCTGAAGCTCAGCAGCATCTGCTGAAGGACACCGTGTGGGGCATCAAGCAGCTGCAGGCTAGGGTGCTGGCAGTCGAGCGCTACCTGCGAGATCAGCAGCTGCTGGGCATCTGGGGGTGTTCCGGAAAGCTGATTTGCTGTACCAATGTGCCATGGAACTCTAGTTGGAGCAATCGCAACCTGTCCGAAATCTGGGACAATATGACTTGGCTGCAGTGGGATAAGGAGATTAGCAACTACACCCAGATCATCTACGGCCTGCTGGAAGAGTCCCAGAATCAGCAGGAGAAGAACGAGCAGGACCTGCTGGCCCTGGAT

# Computational designed hydrophobic core under the variable loops V1,V2, V3. (apolarV1V2+MD39)

BG505_SOSIP_D664_olio6_mC (SEQ ID NO: 37)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFWRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTAPNNFTVKSIRIGPGQAFYYMGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGMFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKLIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 38)GCCGAAAACCTGTGGGTGACTGTCTACTATGGCGTGCCCGTCTGGAAGGACGCAGAGACCACACTGTTCTGCGCTAGCGATGCTAAGGCCTACGAGACCGAGAAACACAACGTGTGGGCAACCCATGCCTGCGTGCCTACAGACCCAAATCCCCAGGAAATCCACCTGGAGAACGTGACCGAGGAGTTCAACATGTGGAAGAACAATATGGTGGAACAGATGCATGAGGACATCATTTCCCTGTGGGATCAGTCTCTGAAGCCCTGCGTGAAACTGACCCCTCTGTGCGTCACACTGCAGTGTACAAACGTGACTAACAATATCACCGACGATATGCGGGGCGAACTGAAGAACTGTTCTTTCAATATGACTACCGAGCTGCGCGACAAGAAACAGAAAGTGTACAGTCTGTTTTGGCGACTGGATGTGGTCCAGATCAACGAAAATCAGGGGAACCGGAGTAACAACTCAAATAAGGAGTATAGACTGATCAACTGCAATACCAGTGCCATTACACAGGCTTGTCCTAAAGTGTCATTCGAGCCTATCCCAATTCATTACTGCGCCCCAGCTGGCTTCGCCATCCTGAAGTGTAAAGATAAGAAGTTCAACGGCACCGGGCCATGCCCTTCAGTGAGCACTGTCCAGTGTACCCACGGAATTAAGCCTGTGGTCTCCACACAGCTGCTGCTGAACGGCTCTCTGGCTGAGGAAGAAGTGATCATTAGATCCGAGAATATCACTAACAATGCAAAGAACATTCTGGTGCAGCTGAATACCCCAGTCCAGATCAACTGCACTGCCCCCAACAATTTCACCGTGAAATCTATCCGGATTGGACCAGGCCAGGCTTTTTACTATATGGGCGACATCATTGGGGATATTAGACAGGCACACTGTAACGTGAGCAAGGCCACATGGAATGAAACTCTGGGGAAGGTGGTCAAACAGCTGCGAAAACATTTCGGAAACAATACAATCATTCGATTTGCACAGAGCAGCGGAGGGGACCTGGAGGTGACAACTCACAGCTTCAACTGCGGAGGCATGTTCTTTTATTGTAATACTAGTGGCCTGTTTAACTCAACTTGGATCAGCAATACCTCCGTGCAGGGCAGCAACAGCACCGGCTCTAATGATAGTATCACACTGCCATGCAGAATTAAGCTGATCATTAATATGTGGCAGAGGATCGGGCAGGCTATGTACGCACCCCCTATCCAGGGAGTGATTCGGTGCGTGAGCAACATCACAGGCCTGATTCTGACTAGAGACGGGGGATCAACAAATAGCACCACAGAGACTTTCAGGCCCGGCGGAGGAGACATGCGAGATAACTGGCGATCCGAACTGTACAAGTATAAAGTGGTCAAGATCGAGCCTCTGGGAGTGGCACCAACCCGATGCAAACGAAGAGTGGTCGGGAGGCGCCGACGGAGAAGGGCTGTGGGGATTGGAGCAGTCTCTCTGGGCTTTCTGGGGGCCGCTGGATCTACAATGGGGGCAGCCAGTATGACTCTGACCGTCCAGGCAAGGAACCTGCTGTCAGGAATCGTGCAGCAGCAGAGCAATCTGCTGCGCGCTCCAGAACCCCAGCAGCACCTGCTGAAGGACACCCATTGGGGCATCAAGCAGCTGCAGGCAAGGGTGCTGGCAGTCGAGCACTACCTGCGAGATCAGCAGCTGCTGGGCATCTGGGGATGCAGCGGAAAGCTGATTTGCTGTACAAACGTGCCCTGGAACAGCAGCTGGAGCAACCGGAATCTGTCCGAAATCTGGGACAACATGACATGGCTGCAGTGGGATAAGGAGATTAGCAATTACACTCAGATCATCTACGGCCTGCTGGAAGAGTCCCAGAACCAGCAGGAGAAGAACGAGCAGGACCTGCTGGCCCTGG AT

# Core mutations from mammalian display data to improve apex bindingbuilt onto olio6

BG505_SOSIP_D664_MD53_mC (SEQ ID NO: 39)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFWRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVLSTQLLLNGSLAEEEVIVRSENITNNAKNILVQLNTPVQINCTAPNNFTVKSIRIGPGQAFYYMGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGMFFFCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKLIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDVWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 40)GCCGAGAACCTGTGGGTGACCGTGTACTATGGCGTGCCCGTGTGGAAGGACGCAGAGACCACACTGTTTTGCGCCAGCGATGCCAAGGCCTATGAGACAGAGAAGCACAACGTGTGGGCAACCCACGCATGCGTGCCTACAGACCCAAATCCCCAGGAGATCCACCTGGAGAACGTGACCGAGGAGTTCAATATGTGGAAGAACAATATGGTGGAGCAGATGCACGAGGACATCATCTCCCTGTGGGATCAGTCTCTGAAGCCCTGCGTGAAGCTGACCCCTCTGTGCGTGACACTGCAGTGTACCAACGTGACAAACAATATCACCGACGATATGCGGGGCGAGCTGAAGAACTGTTCTTTTAATATGACCACAGAGCTGAGGGACAAGAAGCAGAAGGTGTACAGCCTGTTCTGGCGCCTGGATGTGGTGCAGATCAACGAGAATCAGGGCAACAGGTCTAACAATAGCAATAAGGAGTATCGCCTGATCAACTGCAATACCTCCGCCATCACACAGGCCTGTCCTAAGGTGTCTTTTGAGCCTATCCCAATCCACTACTGCGCCCCAGCCGGCTTTGCCATCCTGAAGTGTAAGGATAAGAAGTTCAACGGCACCGGCCCATGCCCTTCCGTGTCTACCGTGCAGTGTACACACGGCATCAAGCCTGTGCTGTCTACACAGCTGCTGCTGAACGGCAGCCTGGCCGAGGAGGAAGTGATCGTGCGGAGCGAGAATATCACCAACAATGCCAAGAACATCCTGGTGCAGCTGAATACACCAGTGCAGATCAACTGCACCGCCCCCAACAACTTCACCGTGAAGTCCATCCGGATCGGCCCAGGCCAGGCCTTCTACTATATGGGCGACATCATCGGCGACATCAGACAGGCCCACTGTAACGTGTCTAAGGCCACCTGGAATGAGACACTGGGCAAGGTGGTGAAGCAGCTGAGGAAGCACTTTGGCAACAATACCATCATCAGGTTCGCACAGAGCAGCGGAGGCGACCTGGAGGTGACCACACACTCCTTTAACTGCGGCGGCATGTTCTTTTTCTGTAATACAAGCGGCCTGTTCAACTCCACCTGGATCTCCAATACATCTGTGCAGGGCAGCAACTCCACCGGCAGCAATGATTCCATCACACTGCCATGCCGGATCAAGCTGATCATCAATATGTGGCAGAGAATCGGCCAGGCCATGTATGCACCACCTATCCAGGGCGTGATCAGATGCGTGAGCAACATCACCGGCCTGATCCTGACAAGAGACGGCGGCTCTACCAATAGCACCACAGAGACCTTCCGGCCCGGCGGAGGCGACATGAGGGACGTGTGGAGATCCGAGCTGTACAAGTATAAGGTGGTGAAGATCGAGCCTCTGGGAGTGGCACCAACCAGGTGCAAGAGGAGAGTGGTGGGCAGGCGCCGGAGAAGGCGCGCAGTGGGCATCGGCGCCGTGTCTCTGGGCTTCCTGGGAGCAGCAGGCAGCACAATGGGCGCAGCCTCTATGACCCTGACAGTGCAGGCCAGGAACCTGCTGAGCGGCATCGTGCAGCAGCAGTCCAATCTGCTGCGCGCCCCAGAGCCACAGCAGCACCTGCTGAAGGACACCCACTGGGGCATCAAGCAGCTGCAGGCCAGAGTGCTGGCCGTGGAGCACTACCTGAGAGATCAGCAGCTGCTGGGCATCTGGGGATGCTCCGGCAAGCTGATCTGCTGTACCAACGTGCCCTGGAACAGCAGCTGGTCTAACCGGAATCTGAGCGAGATCTGGGACAACATGACCTGGCTGCAGTGGGATAAGGAGATCAGCAATTACACACAGATCATCTATGGCCTGCTGGAGGAGTCCCAGAACCAGCAGGAGAAGAATGAGCAGGACCTGCTGGCCCTGG ATBG505_SOSIP_D664_MD37 (SEQ ID NO: 41)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPC C KLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSACTQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTRKSIRIGPGCAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFA Q SSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQCMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAV S LGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQEHLHKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (mutations relative to BG505 SOSIP inbold, underline)

# MD37 has two extra disulfide bonds to stabilize the V3 loop (V120C,Q315C) and to prevent CD4 induced conformational changes (I201C, A433C).

IV: Examples of Trimers with Combined Germline-Targeting Mutations andStabilization Mutations

# This construct includes germline-targeting mutations built on top ofthe MD39 stabilization mutations from III

BG505_SOSIP_D664_MD39_9mut2A_mC (MD39 + 9MUT) (SEQ ID NO: 42)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNYAPFLINNMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRMAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 43)GCCGAGAATCTGTGGGTGACCGTGTACTATGGCGTGCCCGTGTGGAAGGACGCAGAGACCACACTGTTCTGCGCCAGCGATGCCAAGGCCTACGAGACAGAGAAGCACAACGTGTGGGCAACCCACGCATGCGTGCCTACAGACCCAAACCCCCAGGAGATCCACCTGGAGAATGTGACCGAGGAGTTTAACATGTGGAAGAACAATATGGTGGAGCAGATGCACGAGGACATCATCTCCCTGTGGGATCAGTCTCTGAAGCCCTGCGTGAAGCTGACCCCTCTGTGCGTGACCCTGCAGTGTACAAACTATGCCCCCTTCCTGATCAACAATATGAGGGGCGAGCTGAAGAATTGTTCTTTTAACATGACCACAGAGCTGCGGGACAAGAAGCAGAAAGTGTACAGCCTGTTCTATAGACTGGATGTGGTGCAGATCAATGAGAACCAGGGCAATAGGTCTAACAATAGCAACAAGGAGTACCGCCTGATCAATTGCAACACCTCCGCCATCACACAGGCCTGTCCTAAGGTGTCTTTTGAGCCTATCCCAATCCACTATTGCGCCCCAGCCGGCTTCGCCATCCTGAAGTGTAAGGATAAGAAGTTTAACGGCACCGGCCCATGCCCTTCCGTGTCTACCGTGCAGTGTACACACGGCATCAAGCCTGTGGTGTCTACACAGCTGCTGCTGAATGGCAGCCTGGCCGAGGAGGAAGTGATCATCCGGAGCGAGAACATCACCAACAATGCCAAGAATATCCTGGTGCAGCTGAACACACCAGTGCAGATCAATTGCACCAGGCCCAACAATAACACAGTGAAGTCCATCCGCATCGGCCCAGGCCAGGCCTTCTACTATACCGGCGACATCATCGGCGACATCAGAATGGCCCACTGTAACGTGAGCAAGGCCACCTGGAACGAGACACTGGGCAAGGTGGTGAAGCAGCTGCGGAAGCACTTCGGCAATAACACCATCATCAGATTTGCACAGAGCAGCGGAGGCGACCTGGAGGTGACCACACACTCCTTCAATTGCGGCGGCGAGTTCTTTTACTGTAACACAAGCGGCCTGTTTAATTCCACCTGGATCTCCAACACATCTGTGCAGGGCAGCAATTCCACCGGCAGCAACGATTCCATCACACTGCCATGCAGGATCAAGCAGATCATCAACATGTGGCAGAGGATCGGACAGGCAATGTATGCACCACCTATCCAGGGCGTGATCAGATGCGTGAGCAATATCACCGGCCTGATCCTGACACGCGACGGAGGCTCTACCAACAGCACCACAGAGACATTCAGGCCCGGCGGAGGCGACATGAGGGATAACTGGAGATCCGAGCTGTACAAGTATAAGGTGGTGAAGATCGAGCCTCTGGGAGTGGCACCAACCAGGTGCAAGAGGAGAGTGGTGGGCAGGCGCCGGAGAAGGCGCGCAGTGGGCATCGGAGCCGTGTCTCTGGGCTTTCTGGGAGCAGCAGGCTCCACAATGGGCGCAGCCTCTATGACCCTGACAGTGCAGGCCCGGAATCTGCTGAGCGGCATCGTGCAGCAGCAGTCCAACCTGCTGAGAGCCCCAGAGCCCCAGCAGCACCTGCTGAAGGACACCCACTGGGGCATCAAGCAGCTGCAGGCCAGAGTGCTGGCCGTGGAGCACTACCTGAGAGATCAGCAGCTGCTGGGCATCTGGGGATGCTCCGGCAAGCTGATCTGCTGTACCAATGTGCCTTGGAACTCTAGCTGGTCTAATAGAAACCTGAGCGAGATCTGGGACAATATGACCTGGCTGCAGTGGGATAAGGAGATCAGCAACTACACACAGATCATCTATGGCCTGCTGGAGGAGTCCCAGAATCAGCAGGAGAAGAACGAGCAGGACCTGCTGGCCCTGG AT

# This construct includes germline-targeting mutations built on top ofthe MD39 stabilization mutations from III

BG505_SOSIP_D664_MD39_10mut_mC (SEQ ID NO: 44)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNYAPFLINDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYFGDIIGDIRMAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 45)GCCGAGAATCTGTGGGTGACCGTGTACTATGGCGTGCCCGTGTGGAAGGACGCAGAGACCACACTGTTCTGCGCCAGCGATGCCAAGGCCTACGAGACAGAGAAGCACAACGTGTGGGCAACCCACGCATGCGTGCCTACAGACCCAAACCCCCAGGAGATCCACCTGGAGAATGTGACCGAGGAGTTTAACATGTGGAAGAACAATATGGTGGAGCAGATGCACGAGGACATCATCTCCCTGTGGGATCAGTCTCTGAAGCCCTGCGTGAAGCTGACCCCTCTGTGCGTGACCCTGCAGTGTACAAACTATGCCCCCTTCCTGATCAACGATATGAGGGGCGAGCTGAAGAATTGTTCTTTTAACATGACCACAGAGCTGCGGGACAAGAAGCAGAAAGTGTACAGCCTGTTCTATAGACTGGATGTGGTGCAGATCAATGAGAACCAGGGCAATAGGTCTAACAATAGCAACAAGGAGTACCGCCTGATCAATTGCAACACCTCCGCCATCACACAGGCCTGTCCTAAGGTGTCTTTTGAGCCTATCCCAATCCACTATTGCGCCCCAGCCGGCTTCGCCATCCTGAAGTGTAAGGATAAGAAGTTTAACGGCACCGGCCCATGCCCTTCCGTGTCTACCGTGCAGTGTACACACGGCATCAAGCCTGTGGTGTCTACACAGCTGCTGCTGAATGGCAGCCTGGCCGAGGAGGAAGTGATCATCCGGAGCGAGAACATCACCAACAATGCCAAGAATATCCTGGTGCAGCTGAACACACCAGTGCAGATCAATTGCACCAGGCCCAACAATAACACAGTGAAGTCCATCCGCATCGGCCCAGGCCAGGCCTTCTACTATTTTGGCGACATCATCGGCGACATCAGAATGGCCCACTGTAACGTGAGCAAGGCCACCTGGAACGAGACACTGGGCAAGGTGGTGAAGCAGCTGCGGAAGCACTTCGGCAATAACACCATCATCAGATTTGCACAGAGCAGCGGAGGCGACCTGGAGGTGACCACACACTCCTTCAATTGCGGCGGCGAGTTCTTTTACTGTAACACAAGCGGCCTGTTTAATTCCACCTGGATCTCCAACACATCTGTGCAGGGCAGCAATTCCACCGGCAGCAACGATTCCATCACACTGCCATGCAGGATCAAGCAGATCATCAACATGTGGCAGAGGATCGGACAGGCAATGTATGCACCACCTATCCAGGGCGTGATCAGATGCGTGAGCAATATCACCGGCCTGATCCTGACACGCGACGGAGGCTCTACCAACAGCACCACAGAGACATTCAGGCCCGGCGGAGGCGACATGAGGGATAACTGGAGATCCGAGCTGTACAAGTATAAGGTGGTGAAGATCGAGCCTCTGGGAGTGGCACCAACCAGGTGCAAGAGGAGAGTGGTGGGCAGGCGCCGGAGAAGGCGCGCAGTGGGCATCGGAGCCGTGTCTCTGGGCTTTCTGGGAGCAGCAGGCTCCACAATGGGCGCAGCCTCTATGACCCTGACAGTGCAGGCCCGGAATCTGCTGAGCGGCATCGTGCAGCAGCAGTCCAACCTGCTGAGAGCCCCAGAGCCCCAGCAGCACCTGCTGAAGGACACCCACTGGGGCATCAAGCAGCTGCAGGCCAGAGTGCTGGCCGTGGAGCACTACCTGAGAGATCAGCAGCTGCTGGGCATCTGGGGATGCTCCGGCAAGCTGATCTGCTGTACCAATGTGCCTTGGAACTCTAGCTGGTCTAATAGAAACCTGAGCGAGATCTGGGACAATATGACCTGGCTGCAGTGGGATAAGGAGATCAGCAACTACACACAGATCATCTATGGCCTGCTGGAGGAGTCCCAGAATCAGCAGGAGAAGAACGAGCAGGACCTGCTGGCCCTGG AT

# This construct includes germline-targeting mutations built on top ofthe MD39 stabilization mutations from III

BG505_SOSIP_D664_MD39_10mut2A_mC (SEQ ID NO: 46)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNYAPFLINNMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYFGDIIGDIRMAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 47)GCCGAGAATCTGTGGGTGACCGTGTACTATGGCGTGCCCGTGTGGAAGGACGCAGAGACCACACTGTTCTGCGCCAGCGATGCCAAGGCCTACGAGACAGAGAAGCACAACGTGTGGGCAACCCACGCATGCGTGCCTACAGACCCAAACCCCCAGGAGATCCACCTGGAGAATGTGACCGAGGAGTTTAACATGTGGAAGAACAATATGGTGGAGCAGATGCACGAGGACATCATCTCCCTGTGGGATCAGTCTCTGAAGCCCTGCGTGAAGCTGACCCCTCTGTGCGTGACCCTGCAGTGTACAAACTATGCCCCCTTCCTGATCAACAATATGAGGGGCGAGCTGAAGAATTGTTCTTTTAACATGACCACAGAGCTGCGGGACAAGAAGCAGAAAGTGTACAGCCTGTTCTATAGACTGGATGTGGTGCAGATCAATGAGAACCAGGGCAATAGGTCTAACAATAGCAACAAGGAGTACCGCCTGATCAATTGCAACACCTCCGCCATCACACAGGCCTGTCCTAAGGTGTCTTTTGAGCCTATCCCAATCCACTATTGCGCCCCAGCCGGCTTCGCCATCCTGAAGTGTAAGGATAAGAAGTTTAACGGCACCGGCCCATGCCCTTCCGTGTCTACCGTGCAGTGTACACACGGCATCAAGCCTGTGGTGTCTACACAGCTGCTGCTGAATGGCAGCCTGGCCGAGGAGGAAGTGATCATCCGGAGCGAGAACATCACCAACAATGCCAAGAATATCCTGGTGCAGCTGAACACACCAGTGCAGATCAATTGCACCAGGCCCAACAATAACACAGTGAAGTCCATCCGCATCGGCCCAGGCCAGGCCTTCTACTATTTTGGCGACATCATCGGCGACATCAGAATGGCCCACTGTAACGTGAGCAAGGCCACCTGGAACGAGACACTGGGCAAGGTGGTGAAGCAGCTGCGGAAGCACTTCGGCAATAACACCATCATCAGATTTGCACAGAGCAGCGGAGGCGACCTGGAGGTGACCACACACTCCTTCAATTGCGGCGGCGAGTTCTTTTACTGTAACACAAGCGGCCTGTTTAATTCCACCTGGATCTCCAACACATCTGTGCAGGGCAGCAATTCCACCGGCAGCAACGATTCCATCACACTGCCATGCAGGATCAAGCAGATCATCAACATGTGGCAGAGGATCGGACAGGCAATGTATGCACCACCTATCCAGGGCGTGATCAGATGCGTGAGCAATATCACCGGCCTGATCCTGACACGCGACGGAGGCTCTACCAACAGCACCACAGAGACATTCAGGCCCGGCGGAGGCGACATGAGGGATAACTGGAGATCCGAGCTGTACAAGTATAAGGTGGTGAAGATCGAGCCTCTGGGAGTGGCACCAACCAGGTGCAAGAGGAGAGTGGTGGGCAGGCGCCGGAGAAGGCGCGCAGTGGGCATCGGAGCCGTGTCTCTGGGCTTTCTGGGAGCAGCAGGCTCCACAATGGGCGCAGCCTCTATGACCCTGACAGTGCAGGCCCGGAATCTGCTGAGCGGCATCGTGCAGCAGCAGTCCAACCTGCTGAGAGCCCCAGAGCCCCAGCAGCACCTGCTGAAGGACACCCACTGGGGCATCAAGCAGCTGCAGGCCAGAGTGCTGGCCGTGGAGCACTACCTGAGAGATCAGCAGCTGCTGGGCATCTGGGGATGCTCCGGCAAGCTGATCTGCTGTACCAATGTGCCTTGGAACTCTAGCTGGTCTAATAGAAACCTGAGCGAGATCTGGGACAATATGACCTGGCTGCAGTGGGATAAGGAGATCAGCAACTACACACAGATCATCTATGGCCTGCTGGAGGAGTCCCAGAATCAGCAGGAGAAGAACGAGCAGGACCTGCTGGCCCTGG AT

# This construct includes germline-targeting mutations built on top ofthe MD39 stabilization mutations from III

BG505_SOSIP_D664_MD39_11mut2A_mC (SEQ ID NO: 48)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNYAPNLLSNMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYFGDIIGDIRMAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSIVLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 49)GCCGAAAACCTGTGGGTGACTGTCTATTATGGCGTGCCCGTGTGGAAAGATGCTGAAACTACTCTGTTCTGTGCAAGCGATGCTAAGGCCTACGAGACAGAGAAGCACAACGTGTGGGCAACCCACGCATGCGTGCCTACAGACCCAAATCCCCAGGAGATCCACCTGGAGAACGTGACAGAGGAGTTCAATATGTGGAAGAACAATATGGTGGAGCAGATGCACGAGGACATCATCTCCCTGTGGGATCAGTCTCTGAAGCCCTGCGTGAAGCTGACCCCTCTGTGCGTGACCCTGCAGTGTACAAATTATGCCCCAAACCTGCTGTCCAATATGAGGGGCGAGCTGAAGAACTGTTCTTTCAATATGACCACAGAGCTGCGGGACAAGAAGCAGAAGGTGTACAGCCTGTTTTATAGACTGGATGTGGTGCAGATCAATGAGAACCAGGGCAACAGGTCTAACAATAGCAATAAGGAGTACCGCCTGATCAATTGCAACACCAGCGCCATCACACAGGCCTGTCCTAAGGTGTCCTTTGAGCCTATCCCAATCCACTATTGCGCCCCAGCCGGCTTCGCCATCCTGAAGTGTAAGGATAAGAAGTTTAACGGCACCGGCCCATGCCCTTCCGTGTCTACAGTGCAGTGTACACACGGCATCAAGCCAGTGGTGTCTACACAGCTGCTGCTGAACGGCAGCCTGGCCGAGGAGGAAGTGATCATCCGGAGCGAGAATATCACCAACAATGCCAAGAACATCCTGGTGCAGCTGAATACACCAGTGCAGATCAACTGCACCAGGCCCAACAATAACACAGTGAAGTCTATCCGCATCGGCCCCGGCCAGGCCTTCTACTATTTTGGCGACATCATCGGCGATATCAGAATGGCCCACTGTAACGTGAGCAAGGCCACCTGGAATGAGACACTGGGCAAGGTGGTGAAGCAGCTGCGGAAGCACTTCGGCAATAACACCATCATCAGATTTGCACAGTCCTCCGGCGGCGACCTGGAGGTGACCACACACTCCTTCAACTGCGGCGGCGAGTTCTTTTACTGTAATACCAGCGGCCTGTTTAACTCCACCTGGATCTCCAATACATCTGTGCAGGGCAGCAACTCCACAGGCAGCAATGATTCCATCGTGCTGCCCTGCAGGATCAAGCAGATCATCAACATGTGGCAGCGCATCGGCCAGGCCATGTATGCCCCTCCCATCCAGGGCGTGATCAGATGCGTGAGCAACATTACCGGCCTGATCCTGACAAGAGATGGCGGATCTACCAATAGCACAACCGAGACATTCAGGCCCGGCGGCGGCGACATGAGAGATAACTGGAGATCTGAGCTGTACAAGTATAAGGTGGTGAAGATTGAGCCTCTGGGAGTGGCACCAACAAGATGCAAGAGAAGAGTGGTGGGCAGGCGCCGGAGAAGGCGCGCAGTGGGCATTGGAGCCGTGTCCCTGGGCTTTCTGGGAGCAGCAGGATCCACAATGGGAGCAGCCTCTATGACCCTGACAGTGCAGGCCCGGAACCTGCTGAGCGGCATCGTGCAGCAGCAGTCCAATCTGCTGAGAGCCCCAGAGCCCCAGCAGCACCTGCTGAAGGACACCCACTGGGGCATCAAGCAGCTGCAGGCACGGGTGCTGGCAGTGGAGCACTACCTGAGAGATCAGCAGCTGCTGGGCATCTGGGGCTGTAGCGGCAAGCTGATCTGCTGTACCAACGTGCCCTGGAATTCTAGCTGGTCTAATAGAAACCTGAGCGAGATCTGGGACAACATGACCTGGCTGCAGTGGGATAAGGAGATCTCCAATTACACACAGATCATCTATGGCCTGCTGGAGGAATCACAGAATCAGCAGGAAAAGAACGAACAGGATCTGCTGGCACTGG AT

V: Trimers with Modified Surfaces or of Different Strains than BG505,that can be Employed in Strategic Boosting Regimens

# Variable loop cocktails. Shown on MD39 background

BG505_SOSIP_MD39_VLC1-03 (SEQ ID NO: 50)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTDWDNATLANMTGEIKNCSFNMTTELRDKKQKVYSLFYELDIIPIENEYISNNNTSNTSYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSATQWEQTLKGIAAKLLEHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNGSSWNLNKTKENTTNLENGTTTLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGNKSAGIETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 51)GCCGAGAACCTGTGGGTGACCGTGTACTATGGCGTGCCCGTGTGGAAGGACGCAGAGACCACACTGTTCTGCGCCTCCGATGCCAAGGCCTACGAGACAGAGAAGCACAACGTGTGGGCAACCCACGCATGCGTGCCTACAGACCCAAATCCCCAGGAGATCCACCTGGAGAATGTGACAGAGGAGTTTAACATGTGGAAGAACAATATGGTGGAGCAGATGCACGAGGACATCATCTCCCTGTGGGATCAGTCTCTGAAGCCCTGCGTGAAGCTGACCCCTCTGTGCGTGACCCTGCAGTGTACAGACTGGGATAATGCCACCCTGGCCAACATGACAGGCGAGATCAAGAATTGTTCCTTCAACATGACCACAGAGCTGCGGGACAAGAAGCAGAAGGTGTACTCTCTGTTTTATGAGCTGGACATCATCCCCATCGAGAACGAGTACATCAGCAACAATAACACCTCTAATACAAGCTATAGACTGATCAACTGCAATACCTCTGCCATCACACAGGCCTGTCCTAAGGTGAGCTTCGAGCCTATCCCAATCCACTATTGCGCCCCAGCCGGCTTCGCCATCCTGAAGTGTAAGGATAAGAAGTTTAACGGCACCGGCCCATGCCCTAGCGTGTCCACCGTGCAGTGTACACACGGCATCAAGCCTGTGGTGAGCACACAGCTGCTGCTGAACGGCTCCCTGGCCGAGGAGGAAGTGATCATCAGGAGCGAGAATATCACCAATAACGCCAAGAATATCCTGGTGCAGCTGAACACACCAGTGCAGATCAATTGCACCCGGCCCAATAACAATACAGTGAAGTCCATCAGAATCGGCCCAGGCCAGGCCTTTTACTATACCGGCGACATCATCGGCGACATCAGGCAGGCCCACTGTAACGTGTCTGCCACCCAGTGGGAGCAGACACTGAAGGGCATCGCCGCCAAGCTGCTGGAGCACTTCGGCAACAATACCATCATCAGGTTTGCACAGAGCAGCGGAGGCGACCTGGAGGTGACCACACACTCCTTCAATTGCGGCGGCGAGTTCTTTTACTGTAACACCTCTGGCCTGTTTAATGGCTCTAGCTGGAACCTGAATAAGACAAAGGAGAACACCACAAATCTGGAGAACGGCACCATCACACTGCCATGCAGGATCAAGCAGATCATCAACATGTGGCAGAGGATCGGACAGGCAATGTATGCACCACCTATCCAGGGCGTGATCAGATGCGTGAGCAACATCACCGGCCTGATCCTGACAAGAGATGGCGGCAATAAGAGCGCCGGCATCGAGACCTTCCGGCCCGGCGGAGGCGACATGAGGGATAACTGGAGATCCGAGCTGTACAAGTATAAGGTGGTGAAGATCGAGCCTCTGGGCGTGGCCCCAACAAGATGCAAGAGGAGAGTGGTGGGCAGGCGCCGGAGAAGGCGCGCAGTGGGCATCGGAGCCGTGAGCCTGGGCTTTCTGGGAGCAGCAGGCAGCACAATGGGCGCAGCCAGCATGACCCTGACAGTGCAGGCCAGGAACCTGCTGTCCGGCATCGTGCAGCAGCAGTCTAATCTGCTGCGCGCCCCAGAGCCACAGCAGCACCTGCTGAAGGACACCCACTGGGGCATCAAGCAGCTGCAGGCCAGGGTGCTGGCAGTGGAGCACTACCTGAGGGATCAGCAGCTGCTGGGCATCTGGGGATGCAGCGGCAAGCTGATCTGCTGTACCAATGTGCCTTGGAACTCCTCTTGGAGCAACCGGAATCTGTCCGAGATCTGGGACAACATGACCTGGCTGCAGTGGGATAAGGAGATCAGCAATTACACACAGATCATCTATGGCCTGCTGGAGGAGTCCCAGAATCAGCAGGAGAAGAACGAGCAGGACCTGCTGGCCCTGG ATBG505_SOSIP_MD39_VLC2-04 (SEQ ID NO: 52)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCSDYEGNTTRQNITMKEEKGEIKNCSFNMTTELRDKKQKVYSLFYKLDITPIEEDNNSNNSSSANSSNSNANYTNYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSGTKWKNTLKQIVKKLGDHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWNRTNGTWNDVEGLNYTNGNDTITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGNDTDKNETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLL ALD (SEQ ID NO: 53)GCCGAGAACCTGTGGGTGACCGTGTACTATGGCGTGCCCGTGTGGAAGGACGCAGAGACCACACTGTTCTGCGCCTCTGATGCCAAGGCCTACGAGACAGAGAAGCACAACGTGTGGGCAACCCACGCATGCGTGCCTACAGACCCAAACCCCCAGGAGATCCACCTGGAGAACGTGACCGAGGAGTTTAATATGTGGAAGAACAATATGGTGGAGCAGATGCACGAGGACATCATCTCTCTGTGGGATCAGAGCCTGAAGCCCTGCGTGAAGCTGACCCCTCTGTGCGTGACACTGCAGTGTAGCGACTATGAGGGCAACACCACACGGCAGAATATCACCATGAAGGAGGAGAAGGGCGAGATCAAGAACTGTTCTTTCAATATGACCACAGAGCTGAGAGATAAGAAGCAGAAGGTGTACAGCCTGTTTTATAAGCTGGACATCACCCCCATCGAGGAGGATAACAATTCCAACAATAGCTCCTCTGCCAATAGCTCCAATTCTAACGCCAATTACACAAACTATAGGCTGATCAACTGCAATACCTCTGCCATCACACAGGCCTGTCCTAAGGTGAGCTTCGAGCCTATCCCAATCCACTACTGCGCCCCAGCCGGCTTCGCCATCCTGAAGTGTAAGGATAAGAAGTTTAACGGCACCGGCCCATGCCCTAGCGTGTCCACCGTGCAGTGTACACACGGCATCAAGCCTGTGGTGAGCACACAGCTGCTGCTGAATGGCTCCCTGGCCGAGGAGGAAGTGATCATCCGCTCCGAGAACATCACCAACAATGCCAAGAACATCCTGGTGCAGCTGAATACACCAGTGCAGATCAACTGCACCCGGCCCAACAATAACACAGTGAAGTCCATCAGAATCGGCCCAGGCCAGGCCTTTTACTATACCGGCGACATCATCGGCGACATCCGGCAGGCCCACTGTAACGTGAGCGGCACCAAGTGGAAGAACACACTGAAGCAGATCGTGAAGAAGCTGGGCGACCACTTCGGCAATAACACCATCATCAGATTTGCCCAGTCTAGCGGCGGCGATCTGGAGGTGACCACACACAGCTTCAATTGCGGCGGCGAGTTCTTTTACTGTAATACCTCCGGCCTGTTTAACTCTACATGGAACCGGACCAATGGCACATGGAATGACGTGGAGGGCCTGAACTATACCAACGGCAATGATACCATCACACTGCCATGCAGGATCAAGCAGATCATCAATATGTGGCAGAGGATCGGACAGGCAATGTACGCACCACCTATCCAGGGCGTGATCAGATGCGTGAGCAACATCACCGGCCTGATCCTGACAAGAGACGGCGGCAACGACACCGATAAGAATGAGACATTCAGGCCCGGCGGAGGCGACATGAGGGATAACTGGAGATCCGAGCTGTACAAGTATAAGGTGGTGAAGATCGAGCCTCTGGGAGTGGCACCAACCAGGTGCAAGAGGAGAGTGGTGGGCAGGCGCCGGAGAAGGCGCGCAGTGGGCATCGGAGCCGTGTCCCTGGGCTTTCTGGGAGCAGCAGGCTCTACAATGGGCGCAGCCAGCATGACCCTGACAGTGCAGGCCAGGAATCTGCTGTCCGGCATCGTGCAGCAGCAGTCTAACCTGCTGCGCGCCCCAGAGCCACAGCAGCACCTGCTGAAGGACACCCACTGGGGCATCAAGCAGCTGCAGGCCAGGGTGCTGGCAGTGGAGCACTATCTGCGCGATCAGCAGCTGCTGGGCATCTGGGGATGCAGCGGCAAGCTGATCTGCTGTACAAACGTGCCCTGGAACAGCAGCTGGAGCAACAGGAATCTGTCCGAGATCTGGGACAATATGACCTGGCTGCAGTGGGATAAGGAGATCAGCAACTACACACAGATCATCTATGGCCTGCTGGAGGAGTCCCAGAACCAGCAGGAGAAGAATGAGCAGGACCTGCTG GCCCTGGATBG505_SOSIP_MD39_VLC2-08 (SEQ ID NO: 54)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCVTLKNCSNSNCSISRNISIEMDGEIKNCSFNMTTELRDKKQKVYSLFYRLDIVPIESSNNSQLSNNSQVSNNSQSSNYSQYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKKDWEKTLQQVATKLGQHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWIRNSSNSTWNSSASNSTELNSNITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGHETENKTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQ EKNEQDLLALD (SEQ IDNO: 55) GCCGAGAACCTGTGGGTGACAGTGTACTATGGCGTGCCCGTGTGGAAGGACGCAGAGACCACACTGTTCTGCGCCAGCGATGCCAAGGCCTACGAGACCGAGAAGCACAACGTGTGGGCAACACACGCATGCGTGCCTACCGACCCAAATCCCCAGGAGATCCACCTGGAGAATGTGACCGAGGAGTTTAACATGTGGAAGAACAATATGGTGGAGCAGATGCACGAGGACATCATCTCCCTGTGGGATCAGTCTCTGAAGCCCTGCGTGAAGCTGACCCCTCTGTGCGTGACACTGCAGTGCGTGACCCTGAAGAACTGCAGCAATTCCAACTGTTCTATCAGCAGGAATATCTCCATCGAGATGGATGGCGAGATCAAGAATTGTTCTTTCAACATGACCACAGAGCTGCGGGACAAGAAGCAGAAAGTGTACAGCCTGTTCTACCGGCTGGACATCGTGCCCATCGAGAGCAGCAACAATTCTCAGCTGAGCAACAATTCCCAGGTGTCTAACAATAGCCAGTCTAGCAACTACTCCCAGTATCGCCTGATCAATTGCAACACCTCTGCCATCACACAGGCCTGTCCTAAGGTGAGCTTCGAGCCTATCCCAATCCACTATTGCGCCCCAGCCGGCTTCGCCATCCTGAAGTGTAAGGACAAGAAGTTTAACGGCACAGGCCCCTGCCCTTCCGTGTCTACCGTGCAGTGTACACACGGCATCAAGCCTGTGGTGTCTACCCAGCTGCTGCTGAACGGCAGCCTGGCAGAGGAGGAAGTGATCATCCGGAGCGAGAATATCACAAACAATGCCAAGAATATCCTGGTGCAGCTGAACACCCCAGTGCAGATCAATTGCACACGGCCCAACAATAACACCGTGAAGTCCATCAGAATCGGCCCAGGCCAGGCCTTTTACTATACAGGCGACATCATCGGCGACATCCGGCAGGCCCACTGTAACGTGTCTAAGAAGGACTGGGAGAAGACACTGCAGCAGGTGGCCACCAAGCTGGGCCAGCACTTCGGCAATAACACCATCATCAGATTTGCCCAGTCCTCTGGCGGCGATCTGGAGGTGACCACACACTCCTTCAATTGCGGCGGCGAGTTCTTTTACTGTAACACAAGCGGCCTGTTTAATTCCACCTGGATCAGAAATAGCTCCAACTCTACCTGGAACAGCAGCGCCAGCAACTCCACAGAGCTGAATAGCAACATCACCCTGCCATGCAGGATCAAGCAGATCATCAACATGTGGCAGAGGATCGGACAGGCAATGTATGCACCACCTATCCAGGGCGTGATCAGATGCGTGAGCAATATCACAGGCCTGATCCTGACCCGCGATGGAGGACACGAGACCGAGAACAAGACCGAGACATTCAGGCCCGGCGGAGGCGACATGAGGGATAATTGGAGATCCGAGCTGTACAAGTATAAGGTGGTGAAGATCGAGCCTCTGGGAGTGGCACCAACAAGGTGCAAGAGGAGAGTGGTGGGCAGGCGCCGGAGAAGGCGCGCAGTGGGCATCGGAGCCGTGAGCCTGGGCTTTCTGGGAGCAGCAGGCAGCACAATGGGCGCAGCCTCTATGACCCTGACAGTGCAGGCCCGGAACCTGCTGAGCGGCATCGTGCAGCAGCAGTCCAATCTGCTGAGAGCCCCAGAGCCCCAGCAGCACCTGCTGAAGGACACACACTGGGGCATCAAGCAGCTGCAGGCCAGGGTGCTGGCAGTGGAGCACTACCTGAGGGATCAGCAGCTGCTGGGCATCTGGGGATGCTCCGGCAAGCTGATCTGCTGTACCAATGTGCCTTGGAACTCCTCTTGGTCTAATCGGAACCTGAGCGAGATCTGGGACAACATGACATGGCTGCAGTGGGATAAGGAGATCAGCAATTACACCCAGATCATCTATGGCCTGCTGGAGGAGTCCCAGAATCAGCAGGAGAAGAACGAGCAGGACCTGCTGGCCCTGGAT BG505_SOSIP_MD39_VLC3-13 (SEQ ID NO:56) AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNAALTNVTITNGPNITEEIRNCSFNMTTELRDKKQKVYSLFYKLDLVQINGSGGEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSGTKWNETLKQVAGKLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWPENGTMEGSNGTITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGNSTTDTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 57)GCCGAGAACCTGTGGGTGACCGTGTACTATGGCGTGCCAGTGTGGAAGGACGCCGAGACCACACTGTTCTGCGCCTCTGATGCCAAGGCCTACGAGACAGAGAAGCACAACGTGTGGGCAACCCACGCATGCGTGCCAACAGACCCAAACCCCCAGGAGATCCACCTGGAGAATGTGACCGAGGAGTTTAACATGTGGAAGAACAATATGGTGGAGCAGATGCACGAGGACATCATCAGCCTGTGGGATCAGTCCCTGAAGCCCTGCGTGAAGCTGACCCCTCTGTGCGTGACCCTGCAGTGTACAAATGCCGCCCTGACCAACGTGACCATCACAAATGGCCCCAACATCACAGAGGAGATCAGGAATTGTTCCTTCAACATGACCACAGAGCTGCGCGACAAGAAGCAGAAGGTGTACTCTCTGTTTTATAAGCTGGATCTGGTGCAGATCAATGGCAGCGGCGGCGAGTACCGGCTGATCAATTGCAACACCAGCGCCATCACACAGGCCTGTCCTAAGGTGTCCTTCGAGCCTATCCCAATCCACTATTGCGCCCCAGCCGGCTTCGCCATCCTGAAGTGTAAGGACAAGAAGTTTAACGGCACCGGCCCATGCCCTTCCGTGTCTACCGTGCAGTGTACACACGGCATCAAGCCTGTGGTGTCCACACAGCTGCTGCTGAATGGCTCTCTGGCCGAGGAGGAAGTGATCATCAGGAGCGAGAACATCACCAACAATGCCAAGAATATCCTGGTGCAGCTGAACACACCAGTGCAGATCAATTGCACCCGGCCCAACAATAACACAGTGAAGTCTATCAGAATCGGCCCAGGCCAGGCCTTTTACTATACCGGCGACATCATCGGCGACATCCGCCAGGCCCACTGTAATGTGAGCGGCACCAAGTGGAACGAGACACTGAAGCAGGTGGCCGGCAAGCTGAGGAAGCACTTCGGCAATAACACCATCATCCGCTTTGCACAGAGCAGCGGAGGCGATCTGGAGGTGACCACACACTCCTTCAATTGCGGCGGCGAGTTCTTTTACTGTAACACATCTGGCCTGTTTAATAGCACCTGGCCCGAGAACGGCACAATGGAGGGCTCTAATGGCACCATCACACTGCCTTGCCGGATCAAGCAGATCATCAACATGTGGCAGAGAATCGGCCAGGCCATGTATGCACCACCTATCCAGGGCGTGATCAGATGCGTGAGCAATATCACCGGCCTGATCCTGACAAGAGACGGCGGCAACTCCACCACAGATACCGAGACATTCCGGCCCGGCGGAGGCGACATGAGGGATAACTGGAGAAGCGAGCTGTACAAGTATAAGGTGGTGAAGATCGAGCCTCTGGGCGTGGCCCCAACCAGATGCAAGAGGAGAGTGGTGGGCAGGCGCCGGAGAAGGCGCGCAGTGGGCATCGGAGCCGTGTCCCTGGGCTTTCTGGGAGCAGCAGGCAGCACAATGGGCGCAGCCTCCATGACCCTGACAGTGCAGGCCAGGAATCTGCTGTCTGGCATCGTGCAGCAGCAGAGCAACCTGCTGCGCGCCCCAGAGCCACAGCAGCACCTGCTGAAGGACACCCACTGGGGCATCAAGCAGCTGCAGGCCAGGGTGCTGGCAGTGGAGCACTACCTGAGGGATCAGCAGCTGCTGGGCATCTGGGGATGCTCCGGCAAGCTGATCTGCTGTACCAATGTGCCTTGGAACTCTAGCTGGTCCAATAGGAACCTGTCTGAGATCTGGGACAATATGACCTGGCTGCAGTGGGATAAGGAGATCTCCAACTACACACAGATCATCTATGGCCTGCTGGAGGAGTCTCAGAATCAGCAGGAGAAGAACGAGCAGGACCTGCTGGCCCTGGAT BG505_SOSIP_VLC1-03_DS21_mC (SEQ ID NO: 58)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCCKLTPLCVTLQCTDWDNATLANMTGEIKNCSFNMTTELRDKKQKVYSLFYELDIIPIENEYISNNNTSNTSYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGCAFYATGDIIGDIRQAHCNVSATQWEQTLKGIAAKLLEHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNGSSWNLNKTKENTTNLENGTITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGNKSAGIETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 59)GCTGAAAATCTGTGGGTCACTGTGTATTATGGGGTGCCTGTGTGGAAAGACGCTGAGACTACTCTGTTCTGTGCCTCCGACGCTAAAGCCTACGAAACCGAGAAGCACAACGTGTGGGCAACCCATGCCTGCGTCCCTACAGACCCAAATCCCCAGGAAATCCACCTGGAGAATGTGACAGAGGAGTTCAACATGTGGAAAAACAATATGGTGGAGCAGATGCATACTGACATCATTAGCCTGTGGGATCAGTCCCTGAAGCCTTGCTGCAAACTGACCCCACTGTGCGTCACCCTGCAGTGTACAGACTGGGATAATGCTACCCTGGCAAACATGACAGGCGAAATCAAGAATTGTAGTTTCAACATGACCACAGAGCTGAGGGACAAGAAACAGAAAGTGTACTCCCTGTTTTATGAACTGGACATCATTCCCATCGAAAACGAGTACATCTCTAACAACAACACAAGTAACACTTCATATCGCCTGATCAACTGCAATACTAGCGCCATTACCCAGGCTTGTCCAAAGGTGTCCTTTGAGCCTATCCCAATTCATTACTGCGCCCCCGCTGGCTTCGCCATCCTGAAGTGTAAAGATAAGAAGTTCAACGGCACCGGGCCATGCCCTTCCGTGTCTACAGTCCAGTGTACTCACGGAATTAAGCCAGTGGTCAGTACACAGCTGCTGCTGAACGGCTCACTGGCAGAGGAAGAAGTGATGATCCGGAGCGAGAACATCACAAACAACGCTAAGAACATCCTGGTGCAGTTCAACACTCCCGTCCAGATTAATTGCACTAGACCTAACAACAACACCCGGAAAAGCATCAGAATTGGACCCGGCTGCGCCTTTTATGCTACCGGCGACATCATTGGCGACATCCGGCAGGCCCACTGTAACGTGAGCGCTACTCAGTGGGAACAGACCCTGAAGGGGATTGCCGCTAAACTGCTGGAGCATTTCGGAAACAATACCATCATTAGATTTGCCAACAGCTCCGGCGGGGACCTGGAAGTGACTACCCACTCTTTCAATTGCGGAGGCGAGTTCTTTTACTGTAACACTAGTGGACTGTTTAATGGCTCTAGTTGGAACCTGAATAAGACCAAAGAAAACACAACTAATCTGGAGAACGGCACCATCACACTGCCCTGCCGAATTAAGCAGATCATTAACATGTGGCAGCGGATCGGCCAGGCAATGTATGCCCCCCCTATCCAGGGCGTGATCAGATGTGTCTCCAACATCACAGGACTGATTCTGACTAGGGATGGGGGAAACAAGTCTGCCGGGATCGAGACTTTCAGGCCTGGCGGGGGAGACATGAGGGATAACTGGCGCTCCGAACTGTACAAGTATAAAGTGGTCAAGATCGAGCCACTGGGAGTGGCTCCAACACGATGCAAACGAAGAGTGGTCGGAAGGCGACGACGAAGAAGGGCTGTGGGAATCGGAGCAGTCTTCCTGGGCTTTCTGGGGGCAGCCGGATCAACAATGGGCGCTGCAAGCATGACTCTGACCGTGCAGGCACGAAACCTGCTGTCCGGAATCGTCCAGCAGCAGTCTAATCTGCTGCGAGCTCCTGAAGCACAGCAGCATCTGCTGAAGCTGACAGTGTGGGGCATCAAGCAGCTGCAGGCACGGGTGCTGGCAGTCGAGCGCTATCTGCGAGACCAGCAGCTGCTGGGCATCTGGGGGTGTTCTGGAAAGCTGATTTGCTGTACCAATGTGCCCTGGAACAGCAGCTGGTCTAACAGGAATCTGAGTGAAATCTGGGACAACATGACCTGGCTGCAGTGGGATAAGGAGATTTCAAATTACACACAGATCATCTACGGCCTGCTGGAAGAGAGCCAGAATCAGCAGGAGAAGAACGAGCAGGACCTGCTGGCCCTGG ATBG505_SOSIP_VLC2-04_DS21_mC (SEQ ID NO: 60)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCCKLTPLCVTLQCSDYEGNTTRQNITMKEEKGEIKNCSFNMTTELRDKKQKVYSLFYKLDITPIEEDNNSNNSSSANSSNSNANYTNYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGCAFYATGDIIGDIRQAHCNVSGTKWKNTLKQIVKKLGDHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWNRTNGTWNDVEGLNYTNGNDTITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGNDTDKNETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLL ALD (SEQ ID NO: 61)GCCGAAAATCTGTGGGTCACTGTGTATTATGGGGTCCCTGTGTGGAAGGATGCCGAAACTACTCTGTTCTGTGCCTCCGACGCCAAAGCCTACGAAACCGAGAAGCACAATGTGTGGGCCACCCATGCTTGCGTCCCTACAGACCCAAACCCCCAGGAAATCCACCTGGAGAACGTGACAGAGGAGTTCAACATGTGGAAAAACAATATGGTCGAACAGATGCATACTGACATCATTTCCCTGTGGGATCAGTCTCTGAAGCCTTGCTGCAAACTGACACCACTGTGCGTCACTCTGCAGTGTTCCGACTATGAGGGCAACACCACACGGCAGAATATCACCATGAAGGAGGAAAAAGGGGAAATTAAGAACTGTTCATTCAATATGACTACCGAGCTGAGAGATAAGAAACAGAAGGTGTACAGCCTGTTTTATAAACTGGACATCACTCCCATTGAGGAAGATAACAATAGCAACAATAGCTCCTCTGCAAATAGTTCAAATTCCAACGCCAATTACACCAACTATAGGCTGATCAACTGCAATACCAGTGCAATTACACAGGCCTGTCCAAAGGTGTCATTTGAGCCTATCCCAATTCATTACTGCGCTCCCGCAGGCTTCGCCATCCTGAAGTGTAAAGATAAGAAGTTCAACGGCACCGGGCCATGCCCTTCAGTGAGCACTGTCCAGTGTACCCACGGGATCAAGCCAGTGGTCTCCACACAGCTGCTGCTGAATGGATCTCTGGCTGAGGAAGAAGTGATGATCAGGAGCGAGAACATTACCAACAATGCAAAGAACATCCTGGTGCAGTTCAATACACCCGTCCAGATTAACTGCACTCGCCCTAACAATAACACCCGGAAATCCATCAGAATTGGACCCGGCTGCGCATTTTATGCCACCGGCGACATCATTGGCGACATCAGGCAGGCCCACTGTAATGTGTCTGGCACCAAGTGGAAAAACACACTGAAGCAGATTGTGAAGAAACTGGGAGACCATTTCGGCAATAACACAATCATTCGCTTTGCTAATAGCTCCGGCGGGGATCTGGAAGTGACAACTCACAGCTTCAACTGCGGAGGCGAGTTCTTTTACTGTAATACAAGTGGCCTGTTTAACTCAACTTGGAACCGCACAAATGGGACTTGGAATGACGTGGAGGGGCTGAACTATACCAACGGAAATGATACCATCACACTGCCCTGCCGAATTAAGCAGATCATTAATATGTGGCAGCGGATCGGCCAGGCTATGTACGCACCCCCTATCCAGGGCGTGATCAGATGTGTCAGTAACATCACTGGACTGATTCTGACCAGGGACGGGGGAAACGACACAGATAAAAATGAGACTTTCCGGCCTGGCGGGGGAGACATGAGGGATAACTGGCGCTCTGAACTGTACAAGTATAAAGTGGTCAAGATCGAGCCACTGGGAGTGGCCCCCACAAGATGCAAACGAAGAGTGGTCGGAAGGCGACGACGAAGAAGGGCAGTGGGGATCGGAGCTGTCTTCCTGGGCTTTCTGGGGGCCGCTGGATCTACTATGGGAGCAGCCAGTATGACTCTGACCGTGCAGGCTCGCAATCTGCTGTCAGGGATCGTCCAGCAGCAGAGCAACCTGCTGCGAGCCCCTGAAGCTCAGCAGCATCTGCTGAAGCTGACCGTGTGGGGCATCAAGCAGCTGCAGGCTCGGGTGCTGGCAGTCGAGCGCTACCTGCGAGATCAGCAGCTGCTGGGCATCTGGGGGTGTAGCGGAAAGCTGATTTGCTGTACAAACGTGCCCTGGAACAGCAGCTGGAGCAACAGAAATCTGTCCGAAATCTGGGACAATATGACTTGGCTGCAGTGGGATAAGGAGATTTCTAACTACACCCAGATCATCTACGGCCTGCTGGAAGAGAGTCAGAACCAGCAGGAGAAGAACGAGCAGGACCTGCTG GCCCTGGATBG505_SOSIP_VLC2-08_DS21_mC (SEQ ID NO: 62)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCCKLTPLCVTLQCVTLKNCSNSNCSISRNISIEMDGEIKNCSFNMTTELRDKKQKVYSLFYRLDIVPIESSNNSQLSNNSQVSNNSQSSNYSQYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGCAFYATGDIIGDIRQAHCNVSKKDWEKTLQQVATKLGQHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWIRNSSNSTWNSSASNSTELNSNITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGHETENKTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQ EKNEQDLLALD (SEQ IDNO: 63) GCCGAAAATCTGTGGGTCACTGTGTATTATGGCGTGCCCGTGTGGAAGGATGCCGAAACTACTCTGTTCTGTGCCTCCGATGCTAAAGCCTACGAAACCGAGAAGCACAACGTGTGGGCCACTCATGCTTGCGTCCCTACCGACCCAAATCCCCAGGAAATCCACCTGGAGAATGTGACAGAGGAGTTCAACATGTGGAAAAACAATATGGTCGAGCAGATGCATACTGACATCATTAGCCTGTGGGATCAGTCCCTGAAGCCTTGCTGCAAACTGACCCCACTGTGCGTGACACTGCAGTGTGTCACTCTGAAGAACTGCTCTAATAGTAACTGTTCAATCAGCAGGAATATCAGCATTGAAATGGATGGCGAGATTAAGAATTGTTCCTTCAACATGACCACAGAACTGAGAGACAAGAAACAGAAAGTGTACTCCCTGTTCTACCGGCTGGACATCGTCCCCATTGAGAGCTCCAACAATAGCCAGCTGTCCAACAATTCTCAGGTGAGTAACAATTCACAGTCTAGTAACTACTCTCAGTATCGCCTGATCAATTGCAACACTAGTGCAATTACCCAGGCCTGTCCAAAGGTGTCATTTGAGCCTATCCCAATTCATTACTGCGCTCCCGCAGGCTTCGCCATCCTGAAGTGTAAAGACAAGAAGTTCAACGGCACCGGGCCATGCCCTTCCGTGTCTACAGTCCAGTGTACTCACGGCATTAAGCCAGTGGTCAGCACACAGCTGCTGCTGAACGGGTCCCTGGCAGAGGAAGAAGTGATGATCAGGTCTGAGAATATTACCAACAATGCCAAGAATATCCTGGTGCAGTTCAACACACCCGTCCAGATTAATTGCACACGCCCTAACAATAACACTCGGAAATCTATCAGAATTGGACCCGGCTGCGCATTTTATGCCACAGGCGACATCATTGGCGACATCAGGCAGGCCCACTGTAACGTGAGCAAGAAAGACTGGGAGAAGACCCTGCAGCAGGTGGCTACAAAACTGGGACAGCATTTCGGCAATAACACCATCATTCGCTTTGCAAACTCAAGCGGCGGGGATCTGGAAGTGACTACCCACAGCTTCAATTGCGGAGGCGAGTTCTTTTACTGTAACACTTCTGGCCTGTTTAATAGTACCTGGATCAGAAACAGCAGCAACAGCACCTGGAATAGTTCAGCTAGTAACTCAACAGAGCTGAACAGCAACATCACTCTGCCCTGCCGAATTAAGCAGATCATTAACATGTGGCAGCGGATCGGGCAGGCTATGTATGCACCCCCTATCCAGGGAGTGATTCGCTGTGTCAGCAATATCACCGGCCTGATTCTGACACGAGACGGGGGACATGAAACCGAGAACAAAACAGAGACTTTCCGGCCTGGCGGGGGAGACATGAGGGATAATTGGCGCTCCGAACTGTACAAGTATAAAGTGGTCAAGATCGAGCCACTGGGGGTGGCCCCCACTAGATGCAAACGGAGAGTGGTCGGAAGGCGACGACGAAGAAGGGCAGTGGGAATCGGAGCTGTCTTCCTGGGCTTTCTGGGAGCAGCTGGCAGCACAATGGGCGCAGCCTCTATGACCCTGACAGTGCAGGCTCGGAACCTGCTGAGTGGCATCGTCCAGCAGCAGTCAAATCTGCTGAGAGCCCCTGAAGCTCAGCAGCACCTGCTGAAGCTGACAGTGTGGGGCATCAAGCAGCTGCAGGCTCGGGTGCTGGCAGTCGAGCGCTATCTGCGAGATCAGCAGCTGCTGGGCATCTGGGGGTGTAGCGGAAAGCTGATTTGCTGTACTAATGTGCCCTGGAACAGCAGCTGGTCAAATAGAAACCTGAGCGAAATCTGGGACAACATGACTTGGCTGCAGTGGGATAAGGAGATTTCTAATTACACCCAGATCATCTACGGCCTGCTGGAAGAGAGTCAGAATCAGCAGGAGAAGAACGAGCAGGACCTGCTGGCCCTGGAT BG505_SOSIP_VLC3-13_DS21_mC (SEQ IDNO: 64) AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCCKLTPLCVTLQCTNAALTNVTITNGPNITEEIRNCSFNMTTELRDKKQKVYSLFYKLDLVQINGSGGEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGCAFYATGDIIGDIRQAHCNVSGTKWNETLKQVAGKLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWPENGTMEGSNGTITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGNSTTDTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 65)GCCGAAAATCTGTGGGTGACTGTCTATTATGGGGTCCCTGTGTGGAAGGATGCCGAAACTACTCTGTTCTGTGCCAGCGATGCTAAGGCCTACGAAACCGAGAAGCACAATGTGTGGGCAACCCATGCCTGCGTCCCTACAGACCCAAACCCCCAGGAAATCCACCTGGAGAATGTGACCGAGGAGTTCAACATGTGGAAAAACAATATGGTGGAACAGATGCATACAGACATCATTTCCCTGTGGGATCAGTCTCTGAAGCCTTGCTGCAAACTGACCCCACTGTGCGTCACTCTGCAGTGTACCAATGCCGCTCTGACCAACGTGACCATCACAAATGGCCCAAACATCACAGAGGAAATTCGCAATTGTTCTTTCAACATGACCACAGAGCTGCGAGACAAGAAACAGAAGGTGTACAGCCTGTTTTATAAACTGGATCTGGTCCAGATCAATGGGTCCGGCGGGGAATACAGGCTGATCAATTGCAACACAAGTGCTATTACTCAGGCATGTCCAAAGGTGTCATTTGAGCCTATCCCAATTCATTATTGCGCCCCCGCTGGCTTCGCCATCCTGAAGTGTAAAGACAAGAAGTTCAACGGAACTGGCCCCTGCCCTTCAGTGAGCACCGTCCAGTGTACACACGGCATTAAGCCAGTGGTCTCCACCCAGCTGCTGCTGAATGGGTCTCTGGCTGAGGAAGAAGTGATGATCCGGTCCGAGAACATTACTAACAATGCAAAGAATATCCTGGTGCAGTTCAACACCCCCGTCCAGATTAATTGCACTAGACCTAACAATAACACCCGGAAATCCATCAGAATTGGGCCCGGATGCGCTTTTTATGCAACCGGGGACATCATTGGCGACATCCGCCAGGCCCACTGTAATGTGTCTGGCACTAAGTGGAACGAGACCCTGAAACAGGTGGCCGGCAAGCTGCGAAAACATTTCGGGAATAACACAATCATTCGGTTTGCTAATAGCTCCGGAGGCGATCTGGAAGTGACTACCCACAGTTTCAACTGCGGGGGAGAGTTCTTTTACTGTAACACTAGTGGACTGTTTAATTCAACATGGCCTGAAAACGGCACTATGGAGGGCAGCAATGGCACTATCACCCTGCCATGCAGAATTAAGCAGATCATTAACATGTGGCAGAGGATCGGGCAGGCCATGTATGCTCCCCCTATCCAGGGAGTGATTCGGTGTGTCTCAAATATCACAGGCCTGATTCTGACTAGAGACGGCGGGAACAGCACAACTGATACAGAGACTTTCAGGCCCGGAGGCGGGGACATGAGGGATAACTGGCGCAGCGAACTGTACAAGTATAAAGTGGTCAAGATCGAGCCACTGGGAGTGGCACCAACCCGATGCAAACGAAGAGTGGTCGGAAGGCGACGACGAAGAAGGGCAGTGGGCATTGGGGCCGTCTTCCTGGGGTTTCTGGGAGCAGCCGGCTCTACAATGGGAGCTGCAAGTATGACCCTGACAGTGCAGGCTAGGAATCTGCTGTCAGGCATCGTCCAGCAGCAGAGCAACCTGCTGCGAGCACCAGAAGCACAGCAGCATCTGCTGAAGCTGACCGTGTGGGGCATCAAGCAGCTGCAGGCCCGGGTGCTGGCTGTCGAGCGCTACCTGCGAGATCAGCAGCTGCTGGGGATCTGGGGATGTAGCGGCAAGCTGATTTGCTGTACAAATGTGCCTTGGAACTCTAGTTGGAGCAATAGAAACCTGTCCGAAATCTGGGACAATATGACATGGCTGCAGTGGGATAAGGAGATTTCTAACTACACTCAGATCATCTACGGCCTGCTGGAAGAGAGTCAGAATCAGCAGGAGAAGAACGAGCAGGACCTGCTGGCCCTGGAT

# Strain B.US.1998.AC10_029.AY835446 stabilized by olio6 mutations

AC10_SOSIP_olio6_mC (SEQ ID NO: 66)AVEQTWVTVYYGVPVWKEANTTLFCASDAKAYNTEVHNVWATHACVPTDPNPQEVELENVTENFNMWKNNMVDQMHEDIISLWDQSLKPCVKLTPLCVTLSCTDNVGNDTSTNNSRWDKMEKGEIKNCSFNITTNMRDKMQKQYALFWKLDVVPIEEGKNNNSSFTDYRLISCNTSVITQACPKVTFEPIPIHYCAPAGFALLKCKDKKFNGTGPCKNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVVIRSENFSNNARTIIVQLNTSVEIKCIAPNNFTVKGIHIGPGRAFYYMGDIIGDIRQAHCNISRQNWNNTLKQIAEKLREQFGNKTIVFRQSSGGDPEIVMHTFNCAGMFFYCNTAELFNSTWYANGTISIGGGNKTNIILPCRIKLFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGRGNQTDNQTEIFRPVGGDMKNNWRSELYKYKVVRIEPLGIAPTRCKRRVVGRRRRRRAVGIGALSLGFLGAAGSTMGAASMTLTVQARLLLSGIVQQQNNLLRAPEPQQHLLQDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTAVPWNVSWNNRSVDDIWENMTWMQWDREISNYTSLIYTLIEESQNQQEKNEQELLALD (SEQ ID NO: 67)GCCGTGGAGCAGACCTGGGTGACAGTGTACTATGGCGTGCCCGTGTGGAAGGAGGCCAACACCACACTGTTCTGCGCCAGCGACGCCAAGGCCTACAACACCGAGGTGCACAACGTGTGGGCAACCCACGCATGCGTGCCTACAGATCCAAATCCCCAGGAGGTGGAGCTGGAGAACGTGACAGAGAACTTCAACATGTGGAAGAACAATATGGTGGACCAGATGCACGAGGACATCATCTCTCTGTGGGACCAGAGCCTGAAGCCTTGCGTGAAGCTGACCCCACTGTGCGTGACCCTGTCTTGTACAGACAATGTGGGCAACGATACCAGCACAAACAATTCCAGATGGGATAAGATGGAGAAGGGCGAGATCAAGAATTGTAGCTTCAACATCACCACAAATATGAGGGACAAGATGCAGAAGCAGTACGCCCTGTTTTGGAAGCTGGATGTGGTGCCCATCGAGGAGGGCAAGAACAATAACAGCTCCTTCACCGACTATAGACTGATCTCTTGCAATACCAGCGTGATCACACAGGCCTGTCCAAAGGTGACATTTGAGCCTATCCCAATCCACTACTGCGCACCAGCAGGATTCGCACTGCTGAAGTGTAAGGATAAGAAGTTTAATGGCACCGGCCCTTGCAAGAACGTGTCCACCGTGCAGTGTACACACGGCATCAAGCCAGTGGTGTCTACACAGCTGCTGCTGAACGGCAGCCTGGCCGAGGAGGAGGTGGTCATCCGGTCCGAGAATTTCTCTAATAACGCCAGAACCATCATCGTGCAGCTGAACACATCCGTGGAGATCAAGTGCATCGCCCCCAATAACTTCACCGTGAAGGGCATCCACATCGGCCCTGGCAGGGCCTTTTACTATATGGGCGACATCATCGGCGACATCAGGCAGGCCCACTGTAACATCTCTCGCCAGAATTGGAATAACACCCTGAAGCAGATCGCCGAGAAGCTGCGCGAGCAGTTCGGCAATAAGACAATCGTGTTTCGGCAGTCTAGCGGCGGCGACCCAGAGATCGTGATGCACACCTTCAACTGCGCCGGCATGTTCTTTTACTGTAACACAGCCGAGCTGTTTAATTCCACCTGGTATGCCAACGGCACAATCTCTATCGGCGGCGGCAATAAGACCAACATCATCCTGCCCTGCCGCATCAAGCTGTTCATCAATATGTGGCAGGAAGTGGGCAAGGCAATGTACGCACCACCTATCAGCGGACAGATCAGGTGTTCCTCTAACATCACCGGCCTGCTGCTGACACGGGACGGCGGCAGAGGAAACCAGACCGATAATCAGACAGAGATCTTTAGACCTGTGGGCGGCGATATGAAGAATAACTGGCGGTCCGAGCTGTACAAGTATAAGGTGGTGAGAATCGAGCCACTGGGAATCGCACCAACCAGGTGCAAGAGGAGAGTGGTGGGCAGGCGCCGGAGAAGGCGCGCAGTGGGCATCGGCGCCCTGAGCCTGGGCTTTCTGGGAGCAGCAGGCAGCACAATGGGCGCAGCCTCCATGACCCTGACAGTGCAGGCCAGACTGCTGCTGTCCGGCATCGTGCAGCAGCAGAATAACCTGCTGAGGGCCCCCGAGCCTCAGCAGCACCTGCTGCAGGACACCCACTGGGGCATCAAGCAGCTGCAGGCCAGGGTGCTGGCAGTGGAGCACTATCTGCGCGATCAGCAGCTGCTGGGCATCTGGGGCTGCTCTGGCAAGCTGATCTGCTGTACCGCCGTGCCCTGGAACGTGTCCTGGAATAACCGCTCTGTGGACGACATCTGGGAGAATATGACATGGATGCAGTGGGACCGGGAGATCAGCAACTACACCTCCCTGATCTATACACTGATCGAGGAGTCCCAGAACCAGCAGGAGAAGAATGAGCAGGAGCTGCTGGCCCTGGAT

# Resurfaced BG505 SOSIP trimer

BG505_SOSIP_MD39_SET224_wLoops_4_mC (BG505_SOSIP_R4_mC) (SEQ ID NO: 68)AENLWVTVYYGVPVWKDAETTLFCASDAKAYSTEKHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLNCTELNDTNTTATNSSGRVIEDKEIKNCSFNMTTSLRDKVQRVYSLFNKFDIVPIDNSNDSYRLISCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCNDKEFNGTGPCKSVSTVQCTHGIRPVVSTQLLLNGSLAEEEVIIRSENFTNNAKTILVQLNEPVVINCTRPNNNTVKSIRIGPGQAFYYTGEIIGDIRQAHCTVSRETWNKTLGRVVEQLREQFRNKTIIVFNQSSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWYGNETETGGTNDTIGNITLPCRIKQIINMWQEVGKAMYAPPIRGQISCSSNITGLILTRDGGNNNETNTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKCKRRVVGRRRRRRAVGIGAMSLGFLGAAGSTMGAASLTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNTSWSNKSLDQIWDNMTWLEWDREISNYTQLIYNLLEESQNQQEKNEQDLLALD (SEQ ID NO: 69)GCCGAGAATCTGTGGGTGACCGTGTACTATGGCGTGCCCGTGTGGAAGGACGCAGAGACCACACTGTTCTGCGCCTCCGATGCCAAGGCCTACTCTACAGAGAAGCACAACGTGTGGGCAACCCACGCATGCGTGCCTACAGACCCAAATCCCCAGGAGGTGGTGCTGGAGAACGTGACCGAGAACTTTAATATGTGGAAGAACAATATGGTGGAGCAGATGCACGAGGACATCATCTCTCTGTGGGATCAGAGCCTGAAGCCTTGCGTGAAGCTGACCCCACTGTGCGTGACCCTGAATTGTACAGAGCTGAACGACACCAATACCACAGCCACAAACAGCTCCGGCAGAGTGATCGAGGATAAGGAGATCAAGAACTGTAGCTTCAATATGACCACATCCCTGCGGGACAAGGTGCAGAGAGTGTACTCCCTGTTCAATAAGTTTGATATCGTGCCTATCGACAACTCCAATGATTCTTATAGACTGATCAGCTGCAACACCTCCGCCATCACACAGGCCTGTCCAAAGGTGTCTTTTGAGCCTATCCCAATCCACTACTGCGCACCAGCAGGATTCGCAATCCTGAAGTGTAACGACAAGGAGTTTAATGGCACCGGCCCTTGCAAGAGCGTGTCCACCGTGCAGTGTACACACGGCATCCGGCCAGTGGTGTCTACACAGCTGCTGCTGAATGGCAGCCTGGCCGAGGAGGAAGTGATCATCAGAAGCGAGAACTTCACCAACAATGCCAAGACAATCCTGGTGCAGCTGAACGAGCCCGTGGTCATCAACTGCACCAGGCCTAACAATAACACAGTGAAGTCTATCCGCATCGGCCCAGGCCAGGCCTTTTACTATACCGGCGAGATCATCGGCGATATCAGGCAGGCCCACTGTACAGTGAGCCGCGAGACCTGGAACAAGACACTGGGAAGGGTGGTGGAGCAGCTGAGGGAGCAGTTCAGAAATAAGACCATCATCGTGTTTAACCAGTCTAGCGGCGGCGACCCAGAGATCGTGATGCACTCCTTCAACTGCGGCGGCGAGTTCTTTTACTGTAACTCTACCCAGCTGTTTAATAGCACATGGTATGGCAACGAGACCGAGACAGGCGGCACCAACGATACAATCGGCAATATCACCCTGCCCTGCAGGATCAAGCAGATCATCAATATGTGGCAGGAAGTGGGCAAGGCAATGTACGCACCACCTATCAGGGGACAGATCAGCTGTTCCTCTAACATCACCGGCCTGATCCTGACAAGGGACGGAGGCAATAACAATGAGACCAATACCACAGAGACATTCAGACCCGGCGGCGGCGACATGAGGGATAACTGGCGCTCCGAGCTGTACAAGTATAAGGTGGTGAAGATCGAGCCACTGGGAGTGGCACCAACCAAGTGCAAGAGGAGAGTGGTGGGCAGGCGCCGGAGAAGGCGCGCAGTGGGCATCGGCGCCATGAGCCTGGGCTTTCTGGGAGCAGCAGGATCCACAATGGGAGCAGCCTCTCTGACCCTGACAGTGCAGGCCAGGAATCTGCTGAGCGGCATCGTGCAGCAGCAGTCCAACCTGCTGAGGGCACCAGAGCCTCAGCAGCACCTGCTGAAGGACACCCACTGGGGCATCAAGCAGCTGCAGGCACGGGTGCTGGCAGTGGAGCACTATCTGAGAGATCAGCAGCTGCTGGGAATCTGGGGATGCAGCGGCAAGCTGATCTGCTGTACCAACGTGCCTTGGAATACATCTTGGAGCAATAAGTCCCTGGACCAGATCTGGGATAACATGACCTGGCTGGAGTGGGATAGAGAGATCTCCAATTACACACAGCTGATCTATAACCTGCTGGAGGAGTCTCAGAACCAGCAGGAGAAGAATGAGCAGGACCTGCTGGCCC TGGAT

VI: Additional Trimer Modifications that Add Functionality and that canbe Combined with Other Types of Modifications

# Filling conserved glycan holes on BG505: introduction of conservedglycans missing from the BG505 strain: N241 is missing and in 97% of HIVstrains, N289 is missing and in 72% of HIV strains

BG505_SOSIP_MD39_congly_mC (SEQ ID NO: 70)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 71)GCCGAGAACCTGTGGGTGACCGTGTACTATGGCGTGCCCGTGTGGAAGGACGCAGAGACCACACTGTTCTGCGCCAGCGATGCCAAGGCCTACGAGACAGAGAAGCACAACGTGTGGGCAACCCACGCATGCGTGCCTACAGACCCAAACCCCCAGGAGATCCACCTGGAGAATGTGACCGAGGAGTTTAACATGTGGAAGAACAATATGGTGGAGCAGATGCACGAGGACATCATCTCTCTGTGGGATCAGAGCCTGAAGCCTTGCGTGAAGCTGACCCCACTGTGCGTGACACTGCAGTGTACCAACGTGACAAACAATATCACCGACGATATGAGGGGCGAGCTGAAGAATTGTTCTTTCAACATGACCACAGAGCTGCGGGACAAGAAGCAGAAAGTGTACAGCCTGTTTTATAGACTGGATGTGGTGCAGATCAATGAGAACCAGGGCAATAGGAGCAACAATTCCAACAAGGAGTACCGCCTGATCAATTGCAACACCTCTGCCATCACACAGGCCTGTCCTAAGGTGAGCTTCGAGCCTATCCCAATCCACTATTGCGCACCAGCAGGATTCGCAATCCTGAAGTGTAAGGATAAGAAGTTTAATGGCACCGGCCCTTGCCAGAACGTGTCCACCGTGCAGTGTACACACGGCATCAAGCCAGTGGTGAGCACACAGCTGCTGCTGAATGGCTCCCTGGCCGAGGAGGAAGTGATCATCCGGTCTGAGAACATCACCAACAATGCCAAGAATATCCTGGTGCAGCTGAACACAAGCGTGCAGATCAATTGCACCAGGCCCAACAATAACACAGTGAAGTCCATCCGCATCGGCCCTGGCCAGGCCTTTTACTATACCGGCGACATCATCGGCGACATCAGACAGGCCCACTGTAACGTGAGCAAGGCCACCTGGAACGAGACACTGGGCAAGGTGGTGAAGCAGCTGCGGAAGCACTTCGGCAATAACACCATCATCAGATTTGCACAGAGCAGCGGAGGCGACCTGGAGGTGACCACACACTCCTTCAATTGCGGCGGCGAGTTCTTTTACTGTAACACATCCGGCCTGTTTAATTCTACCTGGATCTCTAACACAAGCGTGCAGGGCTCCAATTCTACCGGCTCCAACGATTCTATCACACTGCCATGCAGGATCAAGCAGATCATCAACATGTGGCAGAGGATCGGACAGGCAATGTATGCACCACCTATCCAGGGCGTGATCAGATGCGTGAGCAATATCACCGGCCTGATCCTGACACGCGACGGAGGCAGCACCAACTCCACCACAGAGACATTCAGGCCCGGCGGAGGCGACATGAGGGATAACTGGAGATCCGAGCTGTACAAGTATAAGGTGGTGAAGATCGAGCCACTGGGAGTGGCACCAACCAGGTGCAAGAGGAGAGTGGTGGGCAGGCGCCGGAGAAGGCGCGCAGTGGGCATCGGAGCCGTGAGCCTGGGCTTTCTGGGAGCAGCAGGCTCTACAATGGGCGCAGCCAGCATGACCCTGACAGTGCAGGCCCGGAATCTGCTGTCCGGCATCGTGCAGCAGCAGTCTAACCTGCTGAGAGCCCCCGAGCCTCAGCAGCACCTGCTGAAGGACACCCACTGGGGCATCAAGCAGCTGCAGGCCAGAGTGCTGGCCGTGGAGCACTACCTGAGAGATCAGCAGCTGCTGGGCATCTGGGGATGCTCCGGCAAGCTGATCTGCTGTACCAATGTGCCTTGGAACTCTAGCTGGAGCAATAGAAACCTGTCCGAGATCTGGGACAATATGACCTGGCTGCAGTGGGATAAGGAGATCTCCAACTACACACAGATCATCTATGGCCTGCTGGAGGAGTCTCAGAATCAGCAGGAGAAGAACGAGCAGGACCTGCTGGCCCTGG AT

#C-terminal cysteine constructed used for conjugations. Thismodification can be added to any trimer.

BG505_SOSIP_D664_MD39_CtCys_mC (SEQ ID NO: 72)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDGTKHHHHHHC (SEQ ID NO: 73)GCCGAAAATCTGTGGGTGACTGTCTACTATGGCGTGCCTGTCTGGAAGGACGCCGAGACCACACTGTTCTGTGCTTCCGATGCTAAGGCATACGAAACCGAGAAACACAACGTGTGGGCAACCCATGCCTGCGTCCCAACAGACCCAAACCCCCAGGAAATCCACCTGGAGAATGTGACCGAGGAGTTCAACATGTGGAAGAACAATATGGTGGAACAGATGCATGAGGACATCATTTCCCTGTGGGATCAGTCTCTGAAGCCTTGCGTGAAACTGACCCCACTGTGCGTGACACTGCAGTGTACAAACGTCACTAACAATATCACCGACGATATGCGGGGCGAACTGAAGAATTGTTCTTTCAACATGACTACCGAGCTGAGGGACAAGAAACAGAAAGTGTACAGTCTGTTTTATCGCCTGGATGTGGTCCAGATCAATGAAAACCAGGGGAATCGAAGTAACAATTCAAACAAGGAGTACCGGCTGATTAATTGCAACACCAGTGCCATCACACAGGCTTGTCCAAAAGTGTCATTCGAGCCTATCCCAATTCATTATTGCGCCCCCGCTGGCTTCGCCATCCTGAAGTGTAAAGATAAGAAGTTCAACGGCACCGGGCCATGCCCTTCAGTGAGCACTGTCCAGTGTACCCACGGAATTAAGCCTGTGGTCTCCACACAGCTGCTGCTGAATGGCTCTCTGGCTGAGGAAGAAGTGATCATTAGGTCTGAGAACATCACTAACAATGCAAAGAATATTCTGGTGCAGCTGAACACCCCCGTCCAGATCAATTGCACTCGCCCTAACAATAACACCGTGAAATCTATCCGAATTGGACCCGGCCAGGCTTTTTATTACACCGGCGACATCATTGGCGACATCAGACAGGCACACTGCAATGTGAGCAAGGCCACATGGAACGAGACTCTGGGGAAGGTGGTCAAACAGCTGCGCAAACATTTCGGAAATAACACAATCATTCGATTTGCACAGAGCAGCGGAGGGGACCTGGAAGTGACAACTCACAGCTTCAATTGCGGAGGCGAGTTCTTTTACTGTAACACTAGTGGCCTGTTTAATTCAACTTGGATCAGCAACACCTCCGTGCAGGGCAGCAACAGCACCGGCTCTAACGATAGTATCACACTGCCATGTCGGATTAAGCAGATCATTAACATGTGGCAGAGAATCGGGCAGGCCATGTATGCACCCCCTATCCAGGGAGTGATTCGATGCGTGAGCAATATCACAGGCCTGATTCTGACTAGAGACGGGGGATCAACAAACAGCACCACAGAGACTTTCCGGCCCGGCGGAGGAGACATGCGAGATAACTGGAGATCCGAACTGTACAAGTATAAAGTGGTCAAGATCGAGCCACTGGGCGTGGCTCCCACCAGATGCAAACGAAGAGTGGTCGGGAGGCGCCGACGGAGAAGGGCTGTGGGGATTGGAGCAGTCAGCCTGGGCTTTCTGGGGGCCGCTGGATCTACAATGGGGGCAGCCAGTATGACTCTGACCGTGCAGGCCAGGAATCTGCTGTCAGGAATCGTGCAGCAGCAGAGCAACCTGCTGAGGGCTCCCGAACCCCAGCAGCACCTGCTGAAGGACACCCACTGGGGCATCAAGCAGCTGCAGGCAAGAGTGCTGGCCGTCGAGCATTACCTGAGGGATCAGCAGCTGCTGGGCATCTGGGGATGCAGCGGAAAGCTGATTTGCTGTACAAATGTGCCTTGGAACTCTAGTTGGAGCAATCGCAACCTGTCCGAAATCTGGGACAATATGACATGGCTGCAGTGGGATAAGGAGATTAGCAACTACACTCAGATCATCTACGGCCTGCTGGAAGAGTCCCAGAATCAGCAGGAGAAGAACGAGCAGGACCTGCTGGCCCTGGATGGTACCAAGCACCACCATCACCATCACTGT

# Computational designed mutation knocks out binding of CD4 receptor,yet retains antigenic profile of BG505 CD4bs. In this case Applicantsillustrate the mutation on the MD39 background.

BG505_SOSIP_D664_MD39_CD4KO4_mC (SEQ ID NO: 74)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGTDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 75)GCCGAAAATCTGTGGGTGACTGTCTACTATGGCGTGCCTGTCTGGAAGGACGCCGAGACCACACTGTTCTGTGCTTCCGATGCTAAGGCATACGAAACCGAGAAACACAACGTGTGGGCAACCCATGCCTGCGTCCCAACAGACCCAAACCCCCAGGAAATCCACCTGGAGAATGTGACCGAGGAGTTCAACATGTGGAAGAACAATATGGTGGAACAGATGCATGAGGACATCATTTCCCTGTGGGATCAGTCTCTGAAGCCTTGCGTGAAACTGACCCCACTGTGCGTGACACTGCAGTGTACAAACGTCACTAACAATATCACCGACGATATGCGGGGCGAACTGAAGAATTGTTCTTTCAACATGACTACCGAGCTGAGGGACAAGAAACAGAAAGTGTACAGTCTGTTTTATCGCCTGGATGTGGTCCAGATCAATGAAAACCAGGGGAATCGAAGTAACAATTCAAACAAGGAGTACCGGCTGATTAATTGCAACACCAGTGCCATCACACAGGCTTGTCCAAAAGTGTCATTCGAGCCTATCCCAATTCATTATTGCGCCCCCGCTGGCTTCGCCATCCTGAAGTGTAAAGATAAGAAGTTCAACGGCACCGGGCCATGCCCTTCAGTGAGCACTGTCCAGTGTACCCACGGAATTAAGCCTGTGGTCTCCACACAGCTGCTGCTGAATGGCTCTCTGGCTGAGGAAGAAGTGATCATTAGGTCTGAGAACATCACTAACAATGCAAAGAATATTCTGGTGCAGCTGAACACCCCCGTCCAGATCAATTGCACTCGCCCTAACAATAACACCGTGAAATCTATCCGAATTGGACCCGGCCAGGCTTTTTATTACACCGGCGACATCATTGGCGACATCAGACAGGCACACTGCAATGTGAGCAAGGCCACATGGAACGAGACTCTGGGGAAGGTGGTCAAACAGCTGCGCAAACATTTCGGAAATAACACAATCATTCGATTTGCACAGAGCAGCGGAGGGGACCTGGAAGTGACAACTCACAGCTTCAATTGCGGAGGCGAGTTCTTTTACTGTAACACTAGTGGCCTGTTTAATTCAACTTGGATCAGCAACACCTCCGTGCAGGGCAGCAACAGCACCGGCTCTAACGATAGTATCACACTGCCATGTCGGATTAAGCAGATCATTAACATGTGGCAGAGAATCGGGCAGGCCATGTATGCACCCCCTATCCAGGGAGTGATTCGATGCGTGAGCAATATCACAGGCCTGATTCTGACTAGAGACGGGGGATCAACAAACAGCACCACAGAGACTTTCCGGCCCGGCGGAACCGACATGCGAGATAACTGGAGATCCGAACTGTACAAGTATAAAGTGGTCAAGATCGAGCCACTGGGCGTGGCTCCCACCAGATGCAAACGAAGAGTGGTCGGGAGGCGCCGACGGAGAAGGGCTGTGGGGATTGGAGCAGTCAGCCTGGGCTTTCTGGGGGCCGCTGGATCTACAATGGGGGCAGCCAGTATGACTCTGACCGTGCAGGCCAGGAATCTGCTGTCAGGAATCGTGCAGCAGCAGAGCAACCTGCTGAGGGCTCCCGAACCCCAGCAGCACCTGCTGAAGGACACCCACTGGGGCATCAAGCAGCTGCAGGCAAGAGTGCTGGCCGTCGAGCATTACCTGAGGGATCAGCAGCTGCTGGGCATCTGGGGATGCAGCGGAAAGCTGATTTGCTGTACAAATGTGCCTTGGAACTCTAGTTGGAGCAATCGCAACCTGTCCGAAATCTGGGACAATATGACATGGCTGCAGTGGGATAAGGAGATTAGCAACTACACTCAGATCATCTACGGCCTGCTGGAAGAGTCCCAGAATCAGCAGGAGAAGAACGAGCAGGACCTGCTGGCCCTGG AT

# Computational designed mutation knocks out binding of CD4 receptor,yet retains antigenic profile of BG505 CD4bs. Here the mutation is shownon the olio6 background.

BG505_SOSIP_D664_olio6_CD4KO4_mC (SEQ ID NO: 76)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFWRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTAPNNFTVKSIRIGPGQAFYYMGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGMFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKLIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGTDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (SEQ ID NO: 77)GCCGAAAACCTGTGGGTGACTGTCTACTATGGCGTGCCCGTCTGGAAGGACGCAGAGACCACACTGTTCTGCGCTAGCGATGCTAAGGCCTACGAGACCGAGAAACACAACGTGTGGGCAACCCATGCCTGCGTGCCTACAGACCCAAATCCCCAGGAAATCCACCTGGAGAACGTGACCGAGGAGTTCAACATGTGGAAGAACAATATGGTGGAACAGATGCATGAGGACATCATTTCCCTGTGGGATCAGTCTCTGAAGCCCTGCGTGAAACTGACCCCTCTGTGCGTCACACTGCAGTGTACAAACGTGACTAACAATATCACCGACGATATGCGGGGCGAACTGAAGAACTGTTCTTTCAATATGACTACCGAGCTGCGCGACAAGAAACAGAAAGTGTACAGTCTGTTTTGGCGACTGGATGTGGTCCAGATCAACGAAAATCAGGGGAACCGGAGTAACAACTCAAATAAGGAGTATAGACTGATCAACTGCAATACCAGTGCCATTACACAGGCTTGTCCTAAAGTGTCATTCGAGCCTATCCCAATTCATTACTGCGCCCCAGCTGGCTTCGCCATCCTGAAGTGTAAAGATAAGAAGTTCAACGGCACCGGGCCATGCCCTTCAGTGAGCACTGTCCAGTGTACCCACGGAATTAAGCCTGTGGTCTCCACACAGCTGCTGCTGAACGGCTCTCTGGCTGAGGAAGAAGTGATCATTAGATCCGAGAATATCACTAACAATGCAAAGAACATTCTGGTGCAGCTGAATACCCCAGTCCAGATCAACTGCACTGCCCCCAACAATTTCACCGTGAAATCTATCCGGATTGGACCAGGCCAGGCTTTTTACTATATGGGCGACATCATTGGGGATATTAGACAGGCACACTGTAACGTGAGCAAGGCCACATGGAATGAAACTCTGGGGAAGGTGGTCAAACAGCTGCGAAAACATTTCGGAAACAATACAATCATTCGATTTGCACAGAGCAGCGGAGGGGACCTGGAGGTGACAACTCACAGCTTCAACTGCGGAGGCATGTTCTTTTATTGTAATACTAGTGGCCTGTTTAACTCAACTTGGATCAGCAATACCTCCGTGCAGGGCAGCAACAGCACCGGCTCTAATGATAGTATCACACTGCCATGCAGAATTAAGCTGATCATTAATATGTGGCAGAGGATCGGGCAGGCTATGTACGCACCCCCTATCCAGGGAGTGATTCGGTGCGTGAGCAACATCACAGGCCTGATTCTGACTAGAGACGGGGGATCAACAAATAGCACCACAGAGACTTTCAGGCCCGGCGGAACCGACATGCGAGATAACTGGCGATCCGAACTGTACAAGTATAAAGTGGTCAAGATCGAGCCTCTGGGAGTGGCACCAACCCGATGCAAACGAAGAGTGGTCGGGAGGCGCCGACGGAGAAGGGCTGTGGGGATTGGAGCAGTCTCTCTGGGCTTTCTGGGGGCCGCTGGATCTACAATGGGGGCAGCCAGTATGACTCTGACCGTCCAGGCAAGGAACCTGCTGTCAGGAATCGTGCAGCAGCAGAGCAATCTGCTGCGCGCTCCAGAACCCCAGCAGCACCTGCTGAAGGACACCCATTGGGGCATCAAGCAGCTGCAGGCAAGGGTGCTGGCAGTCGAGCACTACCTGCGAGATCAGCAGCTGCTGGGCATCTGGGGATGCAGCGGAAAGCTGATTTGCTGTACAAACGTGCCCTGGAACAGCAGCTGGAGCAACCGGAATCTGTCCGAAATCTGGGACAACATGACATGGCTGCAGTGGGATAAGGAGATTAGCAATTACACTCAGATCATCTACGGCCTGCTGGAAGAGTCCCAGAACCAGCAGGAGAAGAACGAGCAGGACCTGCTGGCCCTGG AT

# Filling conserved glycan holes on AC10: introduction of conservedglycans missing from the AC10 strain: N295 is missing and in 62% of HIVstrains, N386 is missing and in 86% of HIV strains

AC10_SOSIP_olio6_congly_mC (SEQ ID NO: 78)AVEQTWVTVYYGVPVWKEANTTLFCASDAKAYNTEVHNVWATHACVPTDPNPQEVELENVTENFNMWKNNMVDQMHEDIISLWDQSLKPCVKLTPLCVTLSCTDNVGNDTSTNNSRWDKMEKGEIKNCSFNITTNMRDKMQKQYALFWKLDVVPIEEGKNNNSSFTDYRLISCNTSVITQACPKVTFEPIPIHYCAPAGFALLKCKDKKFNGTGPCKNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVVIRSENFSNNARTIIVQLNTSVEINCTAPNNFTVKGIHIGPGRAFYYMGDIIGDIRQAHCNISRQNWNNTLKQIAEKLREQFGNKTIVFRQSSGGDPEIVMHTFNCAGMFFYCNTSELFNSTWYANGTISIGGGNKTNIILPCRIKLFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGRGNQTDNQTEIFRPVGGDMKNNWRSELYKYKVVRIEPLGIAPTRCKRRVVGRRRRRRAVGIGALSLGFLGAAGSTMGAASMTLTVQARLLLSGIVQQQNNLLRAPEPQQHLLQDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTAVPWNVSWNNRSVDDIWENMTWMQWDREISNYTSLIYTLIEESQNQQEKNEQELLALD (SEQ ID NO: 79)GCCGTGGAGCAGACCTGGGTGACAGTGTACTATGGCGTGCCCGTGTGGAAGGAGGCCAACACCACACTGTTCTGCGCCAGCGACGCCAAGGCCTACAACACCGAGGTGCACAACGTGTGGGCAACCCACGCATGCGTGCCTACAGATCCAAATCCCCAGGAGGTGGAGCTGGAGAACGTGACAGAGAACTTCAACATGTGGAAGAACAATATGGTGGACCAGATGCACGAGGACATCATCTCTCTGTGGGACCAGAGCCTGAAGCCTTGCGTGAAGCTGACCCCACTGTGCGTGACCCTGTCTTGTACAGACAATGTGGGCAACGATACCAGCACAAACAATTCCAGATGGGATAAGATGGAGAAGGGCGAGATCAAGAATTGTAGCTTCAACATCACCACAAATATGAGGGACAAGATGCAGAAGCAGTACGCCCTGTTTTGGAAGCTGGATGTGGTGCCCATCGAGGAGGGCAAGAACAATAACAGCTCCTTCACCGACTATAGACTGATCTCTTGCAATACCAGCGTGATCACACAGGCCTGTCCAAAGGTGACATTTGAGCCTATCCCAATCCACTACTGCGCACCAGCAGGATTCGCACTGCTGAAGTGTAAGGATAAGAAGTTTAATGGCACCGGCCCTTGCAAGAACGTGTCCACCGTGCAGTGTACACACGGCATCAAGCCAGTGGTGTCTACACAGCTGCTGCTGAACGGCAGCCTGGCCGAGGAGGAGGTGGTCATCCGGTCCGAGAATTTCTCTAATAACGCCAGAACCATCATCGTGCAGCTGAACACATCCGTGGAGATCAACTGCACCGCCCCCAATAACTTCACCGTGAAGGGCATCCACATCGGCCCTGGCAGGGCCTTTTACTATATGGGCGACATCATCGGCGACATCAGGCAGGCCCACTGTAACATCTCTCGCCAGAATTGGAATAACACCCTGAAGCAGATCGCCGAGAAGCTGCGCGAGCAGTTCGGCAATAAGACAATCGTGTTTCGGCAGTCTAGCGGCGGCGACCCAGAGATCGTGATGCACACCTTCAACTGCGCCGGCATGTTCTTTTACTGTAACACAAGCGAGCTGTTTAATTCCACCTGGTATGCCAACGGCACAATCTCTATCGGCGGCGGCAATAAGACCAACATCATCCTGCCCTGCCGCATCAAGCTGTTCATCAATATGTGGCAGGAAGTGGGCAAGGCAATGTACGCACCACCTATCAGCGGACAGATCAGGTGTTCCTCTAACATCACCGGCCTGCTGCTGACACGGGACGGCGGCAGAGGAAACCAGACCGATAATCAGACAGAGATCTTTAGACCTGTGGGCGGCGATATGAAGAATAACTGGCGGTCCGAGCTGTACAAGTATAAGGTGGTGAGAATCGAGCCACTGGGAATCGCACCAACCAGGTGCAAGAGGAGAGTGGTGGGCAGGCGCCGGAGAAGGCGCGCAGTGGGCATCGGCGCCCTGAGCCTGGGCTTTCTGGGAGCAGCAGGCAGCACAATGGGCGCAGCCTCCATGACCCTGACAGTGCAGGCCAGACTGCTGCTGTCCGGCATCGTGCAGCAGCAGAATAACCTGCTGAGGGCCCCCGAGCCTCAGCAGCACCTGCTGCAGGACACCCACTGGGGCATCAAGCAGCTGCAGGCCAGGGTGCTGGCAGTGGAGCACTATCTGCGCGATCAGCAGCTGCTGGGCATCTGGGGCTGCTCTGGCAAGCTGATCTGCTGTACCGCCGTGCCCTGGAACGTGTCCTGGAATAACCGCTCTGTGGACGACATCTGGGAGAATATGACATGGATGCAGTGGGACCGGGAGATCAGCAACTACACCTCCCTGATCTATACACTGATCGAGGAGTCCCAGAACCAGCAGGAGAAGAATGAGCAGGAGCTGCTGGCCCTGGAT

Type VII Sequences

191084_SOSIP_MD39 (SEQ ID NO: 80)TENLWVTVYYGVPVWRDAETTLFCASDAKAYDTEMHNVWATHACVPTDPNPQEIDLENVTEKFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLNCTAITNDTRGNETGINRTVETTEMTNCSFNMTTELRDRKKKVNALFYKLDIVQIGENSSSQYRLINCNTSVITQACPKVTFEPIPIHYCAPAGFAILKCKDKEFNGTGTCRNVSSVQCTHGIKPVVSTQLLLNGSLAEGQVIIRSENISDNAKTIIVQLNESVPINCTRPNNNTVRGIHLGPGQTFFYTDIIGDIRQAHCNVSESKWNKALQEVVKQLRQHWNKTIIFKSSSGGDLEITTHSFNCGGEFFYCNTSGLFNSTWNIAGNRTNDTKSNETITLPCRIKQIVNVWQRVGQAIYAPPIAGVIRCNSNITGLLLVRDGGATNNTDETFRPGGGNMRDNWRSELYKYKVVKIEPLGVAPTRCRRRVVERRRRRRAVGLGAVSIGFLGAAGSTMGAASVTLTVQARQLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLKDQQLLGIWGCSGKLICCTTVPWNSSWSNKSQNEIWDNMTWLQWDKEISNYTQLIYSLIEESQNQQEKNEQELLALD 001428_SOSIP_MD39 (SEQ ID NO: 81)VENLWVTVYYGVPVWKEARTTLFCASDAKAYETEVHNVWATHACVPTDPNPQEMVLGNVTENFNMWKNDMVDQMHEDVISLWAQSLKPCVKLTPLCVTLECTQVNATQGNTTQVNVTQVNGDEMKNCSFNTTTEIRDKKQKAYALFYRLDLVPLERENRGDSNSASKYILINCNTSAITQACPKVNFDPIPIHYCTPAGYAILKCNNKTFNGTGSCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIIIRSENLTDNVKTIIVHLDQSVEIVCTRPNNNTVKSIRIGPGQTFYYTGDIIGNIREAHCNISEKKWHEMLRRVSEKLAEHFPNKTIKFTSSSGGDLEITTHSFNCRGEFFYCNTSGLFNSTYMPNGTYMPNGTNNSNSTIILPCRIKQIINMWQEVGPAMYAPPIAGNITCNSNITGLLLVRDGGKNNNTEIFRPGGGDMRDNWRSELYKYKVVEIKPLGVAPTRCKRRVVGRRRRRRAVGLGAVSLGFLGAAGSTMGAASITLTVQARQLLSGIVQQQSNLLQAPEPQQHLLQDTHWGIKQLQTRVLAIEHYLKDQQLLGIWGCSGKLICCTAVPWNSSWSNKSLTDIWDNMTWMQWDREVSNYTGIIYRLLEDSQNQQERNEQDLLALD AC10_SOSIP_MD39 (SEQ ID NO: 82)AVEQTWVTVYYGVPVWKEANTTLFCASDAKAYNTEVHNVWATHACVPTDPNPQEVELENVTENFNMWKNNMVDQMHEDIISLWDQSLKPCVKLTPLCVTLSCTDNVGNDTSTNNSRWDKMEKGEIKNCSFNITTNMRDKMQKQYALFYKLDVVPIEEGKNNNSSFTDYRLISCNTSVITQACPKVTFEPIPIHYCAPAGFALLKCKDKKFNGTGPCKNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVVIRSENFSNNARTIIVQLNTSVEIKCIRPNNNTVKGIHIGPGRAFYYTGDIIGDIRQAHCNISRQNWNNTLKQIAEKLREQFGNKTIVFRQSSGGDPEIVMHTFNCAGEFFYCNTAELFNSTWYANGTISIGGGNKTNIILPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGRGNQTDNQTEIFRPVGGDMKNNWRSELYKYKVVRIEPLGIAPTRCKRRVVGRRRRRRAVGIGALSLGFLGAAGSTMGAASMTLTVQARLLLSGIVQQQNNLLRAPEPQQHLLQDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTAVPWNVSWNNRSVDDIWENMTWMQWDREISNYTSLIYTLIEESQNQQEKNEQELLALD AC10_SOSIP_olio6 (SEQ ID NO:83) AVEQTWVTVYYGVPVWKEANTTLFCASDAKAYNTEVHNVWATHACVPTDPNPQEVELENVTENFNMWKNNMVDQMHEDIISLWDQSLKPCVKLTPLCVTLSCTDNVGNDTSTNNSRWDKMEKGEIKNCSFNITTNMRDKMQKQYALFWKLDVVPIEEGKNNNSSFTDYRLISCNTSVITQACPKVTFEPIPIHYCAPAGFALLKCKDKKFNGTGPCKNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVVIRSENFSNNARTIIVQLNTSVEIKCIAPNNFTVKGIHIGPGRAFYYMGDIIGDIRQAHCNISRQNWNNTLKQIAEKLREQFGNKTIVFRQSSGGDPEIVMHTFNCAGMFFYCNTAELFNSTWYANGTISIGGGNKTNIILPCRIKLFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGRGNQTDNQTEIFRPVGGDMKNNWRSELYKYKVVRIEPLGIAPTRCKRRVVGRRRRRRAVGIGALSLGFLGAAGSTMGAASMTLTVQARLLLSGIVQQQNNLLRAPEPQQHLLQDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTAVPWNVSWNNRSVDDIWENMTWMQWDREISNYTSLIYTLIEESQNQQEKNEQELLALD ZM197M_MD39 (SEQ ID NO: 84)MEQLWVTVYYGVPVWKEAKATLFCASDAKAYEKEVHNVWATHACVPTDPNPQEIPLGNVTENFNMWKNDMADQMHEDIISLWDQSLKPCVKLTPLCVTLNCSDATSNTTKNATNTNTTSTDNRNATSNDTEMKGEIKNCTFNITTEVRDRKTKQRALFYKLDVVPLEEEKNSSSKNSSYKEYRLISCNTSTITQACPKVSFDPIPIHYCAPAGYAILKCNNKTFNGTGPCHNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIIIRSENLTDNTKTIIVHLNESVEINCTRPNNNTVKSVRIGPGQTFFYTGEIIGDIRQAHCNLSKSNWTTTLKRIEKKLKEHFNNATIKFESSAGGDLEITTHSFNCRGEFFYCNTSGLFNSSLLNDTDGTSNSTSNATITLPCRIKQIINMWQEVGRAMYASPIAGIITCKSNITGLLLTRDGGNKSAGIETFRPGGGNMKDNWRSELYKYKVVEIKPLGIAPTSCKRRVVERRRRRRAAGIGAVSLGFLGAAGSTMGAASVMLTVQARQLLSGIVQQQSNLLRAPEPQQHMLQDTHWGIKQLQTRVLAIEHYLKDQQLLGLWGCSGKLICCTAVPWNTSWSNKSKDEIWDNMTWMQWDREIDNYTQVIYQLLEVSQNQQEKNENDLLA LDB41_SOSIP_D664_MD39 (SEQ ID NO: 85)AAKKWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEIVLGNVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLNCNNVNTNNTNNSTNATISDWEKMETGEMKNCSFNVTTSIRDKIKKEYALFYKLDVVPLENKNNINNTNITNYRLINCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNSKTFNGSGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENITDNAKTIIVQLNEAVEINCTRPNNNTVKSIHIGPGRAFYYTGDIIGNIRQAHCNISKARWNETLGQIVAKLEEQFPNKTIIFNHSSGGDPEIVTHSFNCGGEFFYCNTTPLFNSTWNNTRTDDYPTGGEQNITLQCRIKQIINMWQGVGKAMYAPPIRGQIRCSSNITGLLLTRDGGRDQNGTETFRPGGGNMRDNWRSELYKYKVVKIEPLGIAPTACKRRVVQRRRRRRAVGLGAFSLGFLGAAGSTMGAASMALTVQARLLLSGIVQQQNNLLRAPEPQQHMLQDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKIICCTNVPWNDSWSNKTINEIWDNMTWMQWEKEIDNYTQHIYTLLEVSQIQQEKNEQELLELD

Type VIII Sequences

# gp120-gp41 linker optimized by mammalian display directed evolution

BG505_SOSIP_MD39_link14 (SEQ ID NO: 86)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGSHSGSGGSGSGGHAAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD

# The following “CP” designs are all circular permutations of gp140 thateliminate the need for a cleavage site while maintaining native-likestructure.

# Circular permutation 1, variant 1

BG505_SOSIP_MD39_CP1.1 (SEQ ID NO: 87) VSLGFLGAAGSTMGAASM TLTVQARNLLSGIVQQQSNLLRAPEPQQHLLK DT HWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDGGSGSGGGSGSGGSSAENLWVTVYYGVPVWK D AET T LFCAS D AKAYETEKHN VWATHACVPTDPNPQEIHLENVTEEFNMWKN N MVEQMHEDIISLWDQSLKP C VKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYC A PAGFAILKCKD KKFNGTGPCPSVSTVQCTH G IKPVVSTQLLLNGSLAEE E V IIRSENITN NAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYT G DIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVG

# Circular permutation 1, variant 2

BG505_SOSIP_MD39_CP1.2 (SEQ ID NO: 88) VSLGFLGAAGS TMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLK DT HWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLGGSGSGGGSGSGGSSGSGLWVTVYYGVPVWK D AET T LFCAS D AKAYETEKHN V WATHACVPTDPNPQEIHLENVTEEFNMWKN N MVEQMHEDIISLWDQSLKPC V KLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAP A G FAILKCKD KKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEE V II RSENITN NAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTG D IIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRR

# Circular Permutation 2

BG505_SOSIP_MD39_CP2 (SEQ ID NO: 89) VSLGFLGAAGS TMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLK DT HWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTGGSGVTVYYGV PVWK D AET T LFCAS DAKAYETEKHN V WATHACVPTDPNPQEIHLENVTEE FNMWKN N MVEQMHEDIISLWDQSLKPC VKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAP A GFAILKCKD K KFNGTGPCPSV STVQCTH GIKPVVSTQLLLNGSLAEEE V IIRSENITN N AKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTG D IIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRGGSGSGVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD

# Circular Permutation 3

BG505_SOSIP_MD39_CP3 (SEQ ID NO: 90) VSLGFLGAAGS TMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLK DT HWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESGGSGSGAGGLWVTVYYGV PVWK D AET T LFCAS DAKAYETEKHN V WATHACVPTDPNPQEIHLENVTEE FNMWKN N MVEQMHEDIISLWDQSLKPC VKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAP A GFAILKCKD K KFNGTGPCPSV STVQCTH GIKPVVSTQLLLNGSLAEEE V IIRSENITN N AKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTG D IIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGGGSGSGQNQQEKNEQDLLALD

Type IX Sequences

# BG505 MD39 GRSF4 adds glycosylation sites at positions 80, 241, 289,657, and 665 relative to MD39.

BG505_MD39_GRSF4 (SEQ ID NO: 91)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNSSEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNNQSLLALDNGS

# BG505 MD39 GRSF7 adds one additional glycosylation site, at position634, on top of MD39 GRSF4. Adding this glycosylation site isaccomplished by the mutation E634N, but that breaks a salt bridge withR617, so we added the R617A mutation. In total, BG505 MD39 GRSF7contains six extra glycosylation sites relative to BG505 MD39.

BG505_MD39_GRSF7 (SEQ ID NO: 92)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNSSEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNANLSEIWDNMTWLQWDKNISNYTQIIYGLLEESQNQQEKNNQSLLALDNGS

# BG505 CP1.2 GRSF4 adds five glycosylation sites relative to BG505CP1.2, but some of the additional glycosylation sites are different thanthose in BG505_MD39_GRSF4.

BG505_MD39_CP1.2_GRSF4 (SEQ ID NO: 93)VSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLGG N GSGG GSGSGG NGSSGLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNSSEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRR

# Merges the BG505_MD39_link14 modifications with glycan maskingmodifications in BG505 MD39 GRSF4.

BG505_MD39_link14_GRSF4 (SEQ ID NO: 94)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNSSEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGSHSGSGGSGSGGHAAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNNQSLLALDNGS

# Merges the BG505_MD39_link14 modifications with glycan maskingmodifications in BG505 MD39 GRSF7.

BG505_MD39_link14_GRSF7 (SEQ ID NO: 95)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNSSEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGSHSGSGGSGSGGHAAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNANLSEIWDNMTWLQWDKNISNYTQIIYGLLEESQNQQEKNNQSLLALDNGS

Type X Sequences

# Cleavage-independent MD39 with “quiet” loops.

# V1: D141N, R143S (as in VLC1-03), V1 is already short this adds 1extra glycan to c-term of loop

# V2b: Use VLC3-13 (hotspot, single glycan and 8 AA shorter)

# V4: Use VLC3-13 (hotspot, 2 glycans and 5 AA shorter)

BG505_MD39_CP1.2_GRSF4_qLoops1 (SEQ ID NO: 96)VSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLGGNGSGGGSGSGGNGSSGLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNSSEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDNMTGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINGSGGEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWPENGTMEGSNGTITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYK VVKIEPLGVAPTRCKRR

# Same as BG505_MD39_CP1.2_GRSF4_qLoops1, but adds three additionalglycosylation sites (indicated by underline, bold). (This GRSF7 addsmore glycans that BG505 MD39_GRSF7).

BG505_MD39_CP1.2_GRSF7_qLoops1 (SEQ ID NO: 97) GG NSSGSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQ N ESNEQDLGGNGSGGGSGSGGNGSSGLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNSSEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDNMTGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINGSGGEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWPENGTMEGSNGTITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSE LYKYKVVKIEPLGVAPTRC NRS

# Cleavage-dependent BG505_MD39_GRSF4

but with “quiet” loops from BG505_MD39_CP1.2_GRSF4_qLoops1

BG505_MD39_GRSF4_qLoops1 (SEQ ID NO: 98)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNSSEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDNMTGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINGSGGEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWPENGTMEGSNGTITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNNQSLLALDNGS

Type XI Sequences

# BG505 SOSIP MD39 fused via GSGG linker (SEQ ID NO: 116) to thePyrococcus furiosus Ferritin sequence from PDB: 2JD6. Compared to theferritin sequence in 2JD6, the first position in ferritin was mutatedfrom M to G to contribute to the linker. Furthermore, another positionwithin ferritin was mutated from R to K, to eliminate a potential furincleavage site.

BG505_SOSIP_MD39_2JD6 (SEQ ID NO: 99)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDGSGGLSERMLKALNDQLNRELYSAYLYFAMAAYFEDLGLEGFANWMKAQAEEEIGHALRFYNYIYDKNGRVELDEIPKPPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKDYSTRAFLEWFINEQVEEEASVKKILDKLKFAKDSPQILFMLDKELSARAP KLPGLLMQGGE**

# BG505 SOSIP MD39 fused via ASG linker to a variant of DihydrolipoylTransacetylase (E2p) from Bacillus Stearothermophilus. The E2p sequencewas obtained from PDB: 1B5S, and the single unpaired cysteine in thatsequence was either left in place, mutated to A, or mutated to T, asindicated by the “[C/A/T]” expression in the sequence below. Applicantshave found that expression levels are superior when the unpairedcysteine is left intact or when it is mutated to T. The data in FIG. 20correspond to the particle with an A at that position.

BG505 SOSIP MD39 E2p (also referred to as “MD39-1b5s”) (SEQ ID NO: 100)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDASGAAAKPATTEGEFPETREKMSGIRRAIAKAMVHSKHTAPHVTLMDEADVTKLVAHRKKFKAIAAEKGIKLTFLPYVVKALVSALREYPVLNT[C/A/T]IDDETEEIIQKHYYNIGIAADTDRGLLVPVIKHADRKPIFALAQEINELAEKARDGKLTPGEMKGASCTITNIGSAGGQWFTPVINHPEVAILGIGRIAEKPIVRDGEIVAAPMLALSLSFDHRMIDGATAQKALNHIKRLLSDPELLLM**

Type XII Sequences

The first set in Type XII are cleavage-dependent, TM-anchored trimers,some with glycan masking.

#BG505 SOSIP MD39 as gp160 but without cytoplasmic domain

BG505_MD39_gp160_dCT (SEQ ID NO: 101)PQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDKWASLWNWFDISNWLWYIKIFIMIVGGLIGLRIVFAVLSVIHRVRQGYSPLS**

# This is BG505_MD39_gp160_dCT but adds 3 glycoslyation sites (80, 241,289). These 3 additional glycans are a subset of the 5 additionalglycans present in BG505 MD39 GRSF4.

BG505_MD39_gp160_dCT_GRSF4.1 (SEQ ID NO: 102)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNSSEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDKWASLWNWFDISNWLWYIKIFIMIVGGLIGLRIVFAVLSVIHRVRQGYSPLS**

# This is BG505_MD39_gp160_dCT_GRSF4.1 plus the CT domain.

# Full-length gp160 including the Cytoplasmic domain.

BG505_MD39_gp160_GRSF4.1_m (SEQ ID NO: 103)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNSSEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDKWASLWNWFDISNWLWYIKIFIMIVGGLIGLRIVFAVLSVIHRVRQGYSPLSFQTHTPNPRGLDRPERIEEEDGEQDRGRSTRLVSGFLALAWDDLRSLCLFCYHRLRDFILIAARIVELLGHSSLKGLRLGWEGLKYLWNLLAYWGRELKISAINLFDTIAIAVAEWTDRVIEIGQRLCRAFLHIPRRIRQGLERALL**

# Same as BG505_MD39_gp160_dCT but using linker and PDGFR TM domaininstead of native TM domain, for better expression.

BG505_MD39_gp140-PDGFR (SEQ ID NO: 104)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDGGGSGGSGGSEQKLISEEDLGGSGGSGGSNAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIIS LIILIMLWQKKPR**

# Same as BG505_MD39_gp160_dCT_GRSF4.1 but using linker and PDGFR TMdomain instead of native TM domain, for better expression. Same asBG505_MD39_gp140-PDGFR but adding 3 glycans at positions 80, 241, 289.

BG505_MD39_gp140-PDGFR_GRSF4.1_m (SEQ ID NO: 105)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNSSEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDGGGSGGSGGSEQKLISEEDLGGSGGSGGSNAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIIS LIILIMLWQKKPR**

The second set of Type XII sequences are cleavage-independent,TM-anchored trimers, some with glycan masking.

#BG505_MD39_link14 anchored to membrane by linker and PDGFR TM domain

BG505_MD39_gp140-PDGFR_link14 (SEQ ID NO: 106)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGSHSGSGGSGSGGHAAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDGGGSGGSGGSEQKLISEEDLGGSGGSGGSNAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR**

# Cleavage-independent, link14 version of BG505_MD39_gp160_dCT.

BG505_MD39_gp160-dCT_link14 (SEQ ID NO: 107)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGSHSGSGGSGSGGHAAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDKWASLWNWFDISNWLWYIKIFIMIVGGLIGLRIVFAVLSVIHRVRQGYSPLS**

# Same as BG505_MD39_gp160-dCT_link14 but adding 3 glycans, at positions80, 241, 289.

BG505_MD39_gp160-dCT_link14_GRSF4.1 (SEQ ID NO: 108)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNSSEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGSHSGSGGSGSGGHAAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDKWASLWNWFDISNWLWYIKIFIMIVGGLIGLRIVFAVLSVIHRVRQGYSPLS**

# Same as BG505_MD39_gp160-dCT_link14_GRSF4.1 but using linker and PDGFRTM domain instead of native TM domain, for better expression.

BG505_MD39_gp140-PDGFR_link14_GRSF4.1 (SEQ ID NO: 109)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNSSEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGSHSGSGGSGSGGHAAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDGGGSGGSGGSEQKLISEEDLGGSGGSGGSNAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR**

# BG505_MD39_CP1.2_GRSF4 plus linker and PDGFR TM domain.

# Similar to BG505_MD39_gp140-PDGFR_link14_GRSF4.1 but is CP1.2 insteadof link14 and GRSF4.0 instead of GRSF4.1

BG505_MD39_gp140-PDGFR_CP1.2_GRSF4.0 (SEQ ID NO: 110)VSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLGG N GSGG GSGSGG NGSSGLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNSSEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVITHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRGGGSGGSGGSEQKLISEEDLGGSGGSGGSNAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIIL IMLWQKKPR**

# Different Circular permutation than CP1.2; this one uses native gp41C-term so might be better for membrane presentation. Indeed, CP2 doesn'tneed flexible linker to the membrane. This version is gp160 minus thecytoplasmic domain and has additional glycosylation sites added topositions 80, 241, 289.

BG505_MD39_gp160-dCT_CP2_GRSF4.1 (SEQ ID NO: 111) VSLGFLGAAGS TMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLK DT HWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTGGSGVTVYYGV PVWK D AET T LFCAS DAKAYETEKHN V WATHACVPTDPNSSEIHLENVTEE FNMWKN N MVEQMHEDIISLWDQSLKPC VKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAP A GFAILKCKD K KFNGTGPCQNV STVQCTH GIKPVVSTQLLLNGSLAEEE V IIRSENITN N AKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTG D IIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVITHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRGGSGSGVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDKWASLWNWFDISNWLWYIKIFIMIVGGLIGLRIVFAVLSVIHRVRQGYSPLS**

# BG505_MD39_gp160-dCT_CP2_GRSF4.1 but with two extra glycans(NQSLLALDNGS SEQ ID NO: 165)) and with linker and PDGFR TM instead ofnative TM.

BG505_MD39_gp140-PDGFR_CP2_GRSF4.2 (SEQ ID NO: 112) VSLGFLGAAGS TMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLK DT HWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTGGSGVTVYYGV PVWK D AET T LFCAS DAKAYETEKHN V WATHACVPTDPNSSEIHLENVTEE FNMWKN N MVEQMHEDIISLWDQSLKPC VKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAP A GFAILKCKD K KFNGTGPCQNV STVQCTH GIKPVVSTQLLLNGSLAEEE V IIRSENITN N AKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTG D IIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVITHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRGGSGSGVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNNQSLLALDNGSGGGSGGSGGSEQKLISEEDLGGSGGSGGSNAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQ KKPR**

# BG505_MD39_gp160-dCT_CP2_GRSF4.1 but with CT included, so this isfull-length gp160.

BG505_MD39_gp160_CP2_GRSF4.1 (SEQ ID NO: 113) VSLGFLGAAGS TMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLK DT HWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTGGSGVTVYYGV PVWK D AET T LFCAS DAKAYETEKHNVWATHACVPTDPNSSEIHLENVTEE FNMWKN N MVEQMHEDIISLWDQSLKPC VKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAP A GFAILKCKD K KFNGTGPCQNV STVQCTH GIKPVVSTQLLLNGSLAEEE V IIRSENITN N AKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTG D IIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRGGSGSGVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDKWASLWNWFDISNWLWYIKIFIMIVGGLIGLRIVFAVLSVIHRVRQGYSPLSFQTHTPNPRGLDRPERIEEEDGEQDRGRSTRLVSGFLALAWDDLRSLCLFCYHRLRDFILIAARIVELLGHSSLKGLRLGWEGLKYLWNLLAYWGRELKISAINLFDTIAIAVAEWTDRVIEIGQRLCRAFLHIPRRIRQGLERALL**

In one embodiment, the nucleic acids of the present invention may bedelivered as a therapeutic mRNA.

Provided herein are isolated nucleic acids (e.g., modified mRNAsencoding a peptide described herein) comprising a translatable regionand at least two different nucleoside modifications, wherein the nucleicacid exhibits reduced degradation in a cell into which the nucleic acidis introduced, relative to a corresponding unmodified nucleic acid. Forexample, the degradation rate of the nucleic acid is reduced by at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, compared to thedegradation rate of the corresponding unmodified nucleic acid. Incertain embodiments, the nucleic acid comprises RNA, DNA, TNA, GNA, or ahybrid thereof. In certain embodiments, the nucleic acid comprisesmessenger RNA (mRNA). In certain embodiments, the mRNA does notsubstantially induce an innate immune response of the cell into whichthe mRNA is introduced. In certain embodiments, the mRNA comprises atleast one nucleoside selected from the group consisting of pyridin-4-oneribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine,4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine,3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine,1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,1-taurinomethyl-4-thio-uridine, 5-methyl-uridine,1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,dihydropseudouridine, 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine. In certainembodiments, the mRNA comprises at least one nucleoside selected fromthe group consisting of 5-aza-cytidine, pseudoisocytidine,3-methyl-cytidine, N4-acetylcytidine, 5-formyl cytidine,N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine,pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine,2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.In other embodiments, the mRNA comprises at least one nucleosideselected from the group consisting of 2-aminopurine, 2,6-diaminopurine,7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine,7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine,N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine,2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine,7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine. In yetother embodiments, the mRNA comprises at least one nucleoside selectedfrom the group consisting of inosine, 1-methyl-inosine, wyosine,wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine,6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine,6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine,1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine,8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

In some embodiments, the nucleic acids provided herein comprise a 5′untranslated region (UTR) and/or a 3′UTR, wherein each of the twodifferent nucleoside modifications are independently present in the5′UTR and/or 3′UTR. In some embodiments, nucleic acids are providedherein, wherein at least one of the two different nucleosidemodifications are present in the translatable region. In someembodiments, nucleic acids provided herein are capable of binding to atleast one polypeptide that prevents or reduces an innate immune responseof a cell into which the nucleic acid is introduced.

Further provided herein are isolated nucleic acids (e.g., modified mRNAsdescribed herein) comprising (i) a translatable region encoding apeptide described herein, (ii) at least one nucleoside modification, and(iii) at least one intronic nucleotide sequence capable of being excisedfrom the nucleic acid.

Further provided herein are isolated nucleic acids (e.g., modified mRNAsdescribed herein) comprising (i) a translatable region encoding apeptide described herein, (ii) at least two different nucleosidemodifications, and (iii) a degradation domain.

Further provided herein are non-enzymatically synthesized nucleic acids(e.g., modified mRNAs described herein) comprising at least onenucleoside modification, and comprising a translatable region encoding apeptide described herein. In certain embodiments, the non-enzymaticallysynthesized mRNA comprises at least two different nucleosidemodifications.

Further provided herein are isolated nucleic acids (e.g., modified mRNAsdescribed herein) comprising a noncoding region and at least onenucleoside modification that reduces an innate immune response of a cellinto which the nucleic acid is introduced, wherein the nucleic acidsequesters one or more translational machinery components. In certainembodiments, the isolated nucleic acids comprising a noncoding regionand at least one nucleoside modification described herein are providedin an amount effective to reduce protein expression in the cell. Incertain embodiments, the translational machinery component is aribosomal protein or a transfer RNA (tRNA). In certain embodiments, thenucleic acid comprises a small nucleolar RNA (sno-RNA), microRNA(miRNA), small interfering RNA (siRNA) or Piwi-interacting RNA (piRNA).

Further provided herein are isolated nucleic acids (e.g., modified mRNAsdescribed herein) comprising (i) a first translatable region, (ii) atleast one nucleoside modification, and (iii) an internal ribosome entrysite (IRES). In certain embodiments, the IRES is obtained from apicornavirus, a pest virus, a polio virus, an encephalomyocarditisvirus, a foot-and-mouth disease virus, a hepatitis C virus, a classicalswine fever virus, a murine leukemia virus, a simian immune deficiencyvirus or a cricket paralysis virus. In certain embodiments, the isolatednucleic acid further comprises a second translatable region. In certainembodiments, the isolated nucleic acid further comprises a Kozaksequence. In some embodiments, the first translatable region encodes apeptide described herein. In some embodiments, the second translatableregion encodes peptide described herein. In some embodiments, the firstand the second translatable regions encode peptides described herein.

Provided herein are pharmaceutical compositions comprising: (i) aneffective amount of a synthetic messenger ribonucleic acid (mRNA)encoding peptide described herein; and (ii) a pharmaceuticallyacceptable carrier, wherein i) the mRNA comprises pseudouridine, 5′methyl-cytidine, or a combination thereof, or ii) the mRNA does notcomprise a substantial amount of a nucleotide or nucleotides selectedfrom the group consisting of uridine, cytidine, and a combination ofuridine and cytidine, and wherein the composition is suitable forrepeated administration (e.g., intravenous administration) to amammalian subject in need thereof. In some embodiments,

Further provided herein are pharmaceutical compositions comprisingand/or consisting essentially of: (i) an effective amount of a syntheticmessenger ribonucleic acid (mRNA) encoding peptide described herein;(ii) a cell penetration agent; and (iii) a pharmaceutically acceptablecarrier, wherein i) the mRNA comprises pseudouridine, 5′ methyl-cytidineor a combination thereof, or ii) the mRNA does not comprise asubstantial amount of a nucleotide or nucleotides selected from thegroup consisting of uridine, cytidine, and a combination of uridine andcytidine, and wherein the composition is suitable for repeatedadministration (e.g., intravenous administration) to an animal (e.g.,mammalian) subject in need thereof.

This invention provides nucleic acids, including RNAs such as mRNAs thatcontain one or more modified nucleosides (termed “modified nucleicacids”), which have useful properties including the lack of asubstantial induction of the innate immune response of a cell into whichthe mRNA is introduced. Because these modified nucleic acids enhance theefficiency of protein production, intracellular retention of nucleicacids, and viability of contacted cells, as well as possess reducedimmunogenicity, these nucleic acids having these properties are termed“enhanced nucleic acids” herein.

The term “nucleic acid,” in its broadest sense, includes any compoundand/or substance that is or can be incorporated into an oligonucleotidechain. Exemplary nucleic acids for use in accordance with the presentinvention include, but are not limited to, one or more of DNA, RNA,hybrids thereof, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs,miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that inducetriple helix formation, aptamers, vectors, etc., described in detailherein.

Provided are modified nucleic acids containing a translatable regionencoding a peptide described herein, and one, two, or more than twodifferent nucleoside modifications. In some embodiments, the modifiednucleic acid exhibits reduced degradation in a cell into which thenucleic acid is introduced, relative to a corresponding unmodifiednucleic acid. For example, the degradation rate of the nucleic acid isreduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%,compared to the degradation rate of the corresponding unmodified nucleicacid. Exemplary nucleic acids include ribonucleic acids (RNAs),deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycolnucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids(LNAs) or a hybrid thereof. In preferred embodiments, the modifiednucleic acid includes messenger RNAs (mRNAs). As described herein, thenucleic acids of the invention do not substantially induce an innateimmune response of a cell into which the mRNA is introduced.

In some embodiments, modified nucleosides include pyridin-4-oneribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine,4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine,3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine,1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,1-taurinomethyl-4-thio-uridine, 5-methyl-uridine,1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,dihydropseudouridine, 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.

In some embodiments, modified nucleosides include 5-aza-cytidine,pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine,5-formylcytidine, N4-methylcytidine, 5-hydroxymethyl cytidine,1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

In other embodiments, modified nucleosides include 2-aminopurine,2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine,7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine,7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine,1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine,N6-(cis-hydroxyisopentenyl)adenosine,2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine,7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

In certain embodiments it is desirable to intracellularly degrade amodified nucleic acid introduced into the cell, for example if precisetiming of protein production is desired. Thus, the invention provides amodified nucleic acid containing a degradation domain, which is capableof being acted on in a directed manner within a cell.

In other embodiments, modified nucleosides include inosine,1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine,6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine,1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine,8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

Other components of nucleic acid are optional, and are beneficial insome embodiments. For example, a 5′ untranslated region (UTR) and/or a3′UTR are provided, wherein either or both may independently contain oneor more different nucleoside modifications. In such embodiments,nucleoside modifications may also be present in the translatable region.Also provided are nucleic acids containing a Kozak sequence.

Further, nucleic acids encoding a peptide described herein, andcontaining an internal ribosome entry site (IRES) are provided herein.An IRES may act as the sole ribosome binding site, or may serve as oneof multiple ribosome binding sites of an mRNA. An mRNA containing morethan one functional ribosome binding site may encode several peptides orpolypeptides that are translated independently by the ribosomes(“multicistronic mRNA”). When nucleic acids are provided with an IRES,further optionally provided is a second translatable region. Examples ofIRES sequences that can be used according to the invention includewithout limitation, those from picornaviruses (e.g., FMDV), pest viruses(CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV),foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV),classical swine fever viruses (CSFV), murine leukemia virus (MLV),simian immune deficiency viruses (SIV) or cricket paralysis viruses(CrPV).

The therapeutic mRNAs as described, for example, in U.S. Pat. Nos.9,464,124; 9,447,164; 9,428,535; 9,334,328; 9,303,079; 9,301,993;9,295,689; 9,283,287; 9,271,996; 9,255,129; 9,254,311; 9,233,141;9,221,891; 9,220,792; 9,220,755; 9,216,205; 9,192,651; 9,186,372;9,181,319; 9,149,506; 9,114,113; 9,107,886; 9,095,552; 9,089,604;9,061,059; 9,050,297; 8,999,380; 8,980,864; 8,822,663; 8,754,062;8,710,200; 8,680,069 and 8,664,194 may be utilized for the presentinvention.

Methods for the chemical conjugation of polypeptides, carbohydrates,and/or lipids are well known in the art (see, for example, Hermanson.Bioconjugate Techniques (Academic Press; 1992); Aslam and Dent, eds.Bioconjugation: Protein coupling Techniques for the Biomedical Sciences(MacMillan: 1998); and Wong Chemistry of Protein Conjugation andCross-linking (CRC Press: 1991)). For instance, primary amino groups maybe incorporated by reaction with ethylenediamine in the presence ofsodium cyanoborohydride and sulfhydryls may be introduced by reaction ofcysteamin dihydrochloride followed by reduction with a standarddisulfide reducing agent. Heterobifunctional crosslinkers, such as, forexample, sulfosuccinimidyl (4-iodoacetyl) aminobenzoate, which link theepsilon amino group on the D-lysine residues of copolymers of D-lysineand D-glutamate to a sulfhydryl side chain from an amino terminalcysteine residue on the peptide to be coupled, may be used as well.Chemical conjugation also includes anything covalently bonded directlyvia side chain bonds or via a linker or spacer group.

The nanoparticle formulations may be a carbohydrate nanoparticlecomprising a carbohydrate carrier and a modified nucleic acid molecule(e.g., mmRNA). As a non-limiting example, the carbohydrate carrier mayinclude, but is not limited to, an anhydride-modified phytoglycogen orglycogen-type material, phtoglycogen octenyl succinate, phytoglycogenbeta-dextrin, anhydride-modified phytoglycogen beta-dextrin. (See e.g.,International Publication No. WO2012109121; herein incorporated byreference in its entirety).

Lipid nanoparticle formulations may be improved by replacing thecationic lipid with a biodegradable cationic lipid which is known as arapidly eliminated lipid nanoparticle (reLNP). Ionizable cationiclipids, such as, but not limited to, DLinDMA, DLin-KC2-DMA, andDLin-MC3-DMA, have been shown to accumulate in plasma and tissues overtime and may be a potential source of toxicity. The rapid metabolism ofthe rapidly eliminated lipids can improve the tolerability andtherapeutic index of the lipid nanoparticles by an order of magnitudefrom a 1 mg/kg dose to a 10 mg/kg dose in rat. Inclusion of anenzymatically degraded ester linkage can improve the degradation andmetabolism profile of the cationic component, while still maintainingthe activity of the reLNP formulation. The ester linkage can beinternally located within the lipid chain or it may be terminallylocated at the terminal end of the lipid chain. The internal esterlinkage may replace any carbon in the lipid chain.

The average diameter of the nanoparticle employed in the compositions ofthe invention can be at least one member selected from the groupconsisting of about 20 nanometers, about 25 nanometers, about 30nanometers, about 40 nanometers, about 50 nanometers, about 75nanometers, about 100 nanometers, about 125 nanometers, about 150nanometers, about 175 nanometers and about 200 nanometers. In anotherembodiment, the average diameter of the particle is at least one memberselected from the group consisting of between about 10 to about 200nanometers, between about 0.5 to about 5 microns and between about 5 toabout 10 microns. In another embodiment, the average diameter of themicroparticle is selected from the group consisting of about 0.1 m,about 0.2 m, about 0.4 m, about 0.5 m, about 1 m and about 2 m.

Nanoparticles for use in the compositions of the invention can be madefrom lipids or other fatty acids (see, for example, U.S. Pat. Nos.5,709,879; 6,342,226; 6,090,406; Lian, et al., J. of Pharma. Sci.90:667-680 (2001) and van Slooten, et al., Pharm Res. 17:42-48 (2000))and non-lipid compositions (see, for example, Kreuter, J. Anat.189:503-505 (1996), the teachings of all of which are herebyincorporated by reference in their entirety). The compositions can bebilayer or multilamellar liposomes and phospholipid based. Polymerizednanoparticles, as described, for example, in U.S. Pat. No. 7,285,289,the teachings of which are incorporated by reference in their entirety.

Metallic oxide nanoparticles for use in the compositions of theinvention can be chemically substituted with at least one reactivemoiety capable of forming a thioether bond employing conventionallytechniques as described herein and in U.S. Pat. No. 6,086,881, theteachings of which are hereby incorporated by reference in theirentirety. The antigen described herein can be coupled in a single steponto the metallic oxide particles by the formation of at least onethioether bond or it may be synthesized or assembled stepwise onto themetallic oxide particles after the initial thioether bond formation. Thechemical derivatization reagents for the metallic oxide particles caninclude organosilane reagents that provide thioalkane functionality orother groups that may readily be converted into thiols or thiol-reactivemoieties. Organosilane reagents which may be utilized for this purposemay be, but are not limited to, 3-mercaptopropyltrimethoxysilane,3-aminopropyltriethoxysilane, 3-iodopropyltrimethoxysilane,2-chloroethyltrichlorosilane, 3-glycidoxypropyltrimethoxysilane,vinyltrichlorosilane and 3-acryloxypropyltrimethoxysilane. Moieties thatinclude one or more disulfide components may also be joined to themetallic oxide particle surface and thereby provide the correspondingreactive moiety able to enter into and form a thioether bond andjuncture. Exemplary nanoparticles for use in the compositions of theinvention include at least one member selected from the group consistingof poly (d,l-lactide-co-glycolide, also referred to as“poly(lactic-co-glycolic acid) and bisacyloxypropylcysteine.

Nanoparticles for use in the compositions of the invention can be madeof inorganic material. Nanoparticles for use in the compositions of theinvention can be made of a polymer material, such as at least one memberselected from the group consisting of polystyrene, brominatedpolystyrene, polyacrylic acid, polyacrylonitrile, polyamide,polyacrylamide, polyacrolein, polybutadiene, polycaprolactone,polycarbonate, polyester, polyethylene, polyethylene terephthalate,polydimethylsiloxane, polyisoprene, polyurethane, polyvinylacetate,polyvinylchloride, polyvinylpyridine, polyvinylbenzylchloride,polyvinyltoluene, polyvinylidene chloride, polydivinylbenzene,polymethylmethacrylate, polylactide, polyglycolide,poly(lactide-co-glycolide), polyanhydride, polyorthoester,polyphosphazene, polyphosophaze, a carbohydrate, carboxymethylcellulose, hydroxyethyl cellulose, agar, gel, proteinaceous polymer,polypeptide, eukaryotic and prokaryotic cells, viruses, lipid, metal,resin, latex, rubber, silicone (e.g., polydimethyldiphenyl siloxane),glass, ceramic, charcoal, kaolinite and bentonite.

It is noted that these therapeutics may be a chemical compound, acomposition which may comprise a polypeptide of the present inventionand/or antibody elicited by such a chemical compound and/or portionthereof or a pharmaceutically acceptable salt or a composition which maycomprise a polypeptide of the invention, and may be administered aloneor as an active ingredient in combination with pharmaceuticallyacceptable carriers, diluents, and vehicles, as well as other activeingredients.

The compounds or compositions may be administered orally, subcutaneouslyor parenterally including intravenous, intraarterial, intramuscular,intraperitoneally, and intranasal administration as well as intrathecaland infusion techniques.

It is noted that humans are treated generally longer than the mice orother experimental animals which treatment has a length proportional tothe length of the disease process and drug effectiveness. The doses maybe single doses or multiple doses over a period of several days, butsingle doses are preferred. Thus, one may scale up from animalexperiments, e.g., rats, mice, and the like, to humans, by techniquesfrom this disclosure and documents cited herein and the knowledge in theart, without undue experimentation.

The treatment generally has a length proportional to the length of thedisease process and drug effectiveness and the patient being treated.

When administering a therapeutic of the present invention parenterally,it will generally be formulated in a unit dosage injectable form(solution, suspension, emulsion). The pharmaceutical formulationssuitable for injection include sterile aqueous solutions or dispersionsand sterile powders for reconstitution into sterile injectable solutionsor dispersions. The carrier may be a solvent or dispersing mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol, and the like), suitablemixtures thereof, and vegetable oils.

Proper fluidity may be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Nonaqueousvehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, cornoil, sunflower oil, or peanut oil and esters, such as isopropylmyristate, may also be used as solvent systems for compoundcompositions.

Additionally, various additives which enhance the stability, sterility,and isotonicity of the compositions, including antimicrobialpreservatives, antioxidants, chelating agents, and buffers, may beadded. Prevention of the action of microorganisms may be ensured byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, and the like. In many cases, it willbe desirable to include isotonic agents, for example, sugars, sodiumchloride, and the like. Prolonged absorption of the injectablepharmaceutical form may be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin. According tothe present invention, however, any vehicle, diluent, or additive usedwould have to be compatible with the compounds.

Sterile injectable solutions may be prepared by incorporating thecompounds utilized in practicing the present invention in the requiredamount of the appropriate solvent with various amounts of the otheringredients, as desired.

A pharmacological formulation of the present invention, e.g., which maycomprise a therapeutic compound or polypeptide of the present invention,may be administered to the patient in an injectable formulationcontaining any compatible carrier, such as various vehicles, adjuvants,additives, and diluents; or the compounds utilized in the presentinvention may be administered parenterally to the patient in the form ofslow-release subcutaneous implants or targeted delivery systems such asmonoclonal antibodies, iontophoretic, polymer matrices, liposomes, andmicrospheres.

A pharmacological formulation of the compound and composition which maycomprise a polypeptide utilized in the present invention may beadministered orally to the patient. Conventional methods such asadministering the compounds in tablets, suspensions, solutions,emulsions, capsules, powders, syrups and the like are usable. Knowntechniques, which deliver the compound orally or intravenously andretain the biological activity, are preferred.

In one embodiment, a formulation of the present invention may beadministered initially, and thereafter maintained by furtheradministration. For instance, a formulation of the invention may beadministered in one type of composition and thereafter furtheradministered in a different or the same type of composition. Forexample, a formulation of the invention may be administered byintravenous injection to bring blood levels to a suitable level. Thepatient's levels are then maintained by an oral dosage form, althoughother forms of administration, dependent upon the patient's condition,may be used. In the instance of a vaccine composition, the vaccine maybe administered as a single dose, or the vaccine may incorporate setbooster doses. For example, booster doses may comprise variants in orderto provide protection against multiple clades of HIV.

The quantity to be administered will vary for the patient being treatedand whether the administration is for treatment or prevention and willvary from a few micrograms to a few milligrams for an average 70 kgpatient, e.g., 5 micrograms to 5 milligrams such as 500 micrograms, orabout 100 ng/kg of body weight to 100 mg/kg of body weight peradministration and preferably will be from 10 pg/kg to 10 mg/kg peradministration. Typically, however, the antigen is present in an amounton, the order of micrograms to milligrams, or, about 0.001 to about 20wt %, preferably about 0.01 to about 10 wt %, and most preferably about0.05 to about 5 wt %.

Of course, for any composition to be administered to an animal or human,including the components thereof, and for any particular method ofadministration, it is preferred to determine therefor: toxicity, such asby determining the lethal dose (LD) and LD₅₀ in a suitable animal modele.g., rodent such as mouse; and, the dosage of the composition(s),concentration of components therein and timing of administering thecomposition(s), which elicit a suitable immunological response, such asby titrations of sera and analysis thereof for antibodies or antigens,e.g., by ELISA and/or RFFIT analysis. Such determinations do not requireundue experimentation from the knowledge of the skilled artisan, thisdisclosure and the documents cited herein. And, the time for sequentialadministrations may be ascertained without undue experimentation. Forinstance, dosages may be readily ascertained by those skilled in the artfrom this disclosure and the knowledge in the art. Thus, the skilledartisan may readily determine the amount of compound and optionaladditives, vehicles, and/or carrier in compositions and to beadministered in methods of the invention. Typically, an adjuvant oradditive is commonly used as 0.001 to 50 wt % solution in phosphatebuffered saline, and the active ingredient is present in the order ofmicrograms to milligrams, such as about 0.0001 to about 5 wt %,preferably about 0.0001 to about 1 wt %, most preferably about 0.0001 toabout 0.05 wt % or about 0.001 to about 20 wt %, preferably about 0.01to about 10 wt %, and most preferably about 0.05 to about 5 wt %. Suchdeterminations do not require undue experimentation from the knowledgeof the skilled artisan, this disclosure and the documents cited herein.And, the time for sequential administrations may be ascertained withoutundue experimentation.

Examples of compositions which may comprise a therapeutic of theinvention include liquid preparations for orifice, e.g., oral, nasal,anal, vaginal, peroral, intragastric, mucosal (e.g., perlingual,alveolar, gingival, olfactory or respiratory mucosa) etc.,administration such as suspensions, syrups or elixirs; and, preparationsfor parenteral, subcutaneous, intradermal, intramuscular or intravenousadministration (e.g., injectable administration), such as sterilesuspensions or emulsions. Such compositions may be in admixture with asuitable carrier, diluent, or excipient such as sterile water,physiological saline, glucose or the like. The compositions may also belyophilized. The compositions may contain auxiliary substances such aswetting or emulsifying agents, pH buffering agents, gelling or viscosityenhancing additives, preservatives, flavoring agents, colors, and thelike, depending upon the route of administration and the preparationdesired. Standard texts, such as “REMINGTON'S PHARMACEUTICAL SCIENCE”,17th edition, 1985, incorporated herein by reference, may be consultedto prepare suitable preparations, without undue experimentation.

Compositions of the invention, are conveniently provided as liquidpreparations, e.g., isotonic aqueous solutions, suspensions, emulsionsor viscous compositions which may be buffered to a selected pH. Ifdigestive tract absorption is preferred, compositions of the inventionmay be in the “solid” form of pills, tablets, capsules, caplets and thelike, including “solid” preparations which are time-released or whichhave a liquid filling, e.g., gelatin covered liquid, whereby the gelatinis dissolved in the stomach for delivery to the gut. If nasal orrespiratory (mucosal) administration is desired, compositions may be ina form and dispensed by a squeeze spray dispenser, pump dispenser oraerosol dispenser. Aerosols are usually under pressure by means of ahydrocarbon. Pump dispensers may preferably dispense a metered dose or,a dose having a particular particle size.

Compositions of the invention may contain pharmaceutically acceptableflavors and/or colors for rendering them more appealing, especially ifthey are administered orally. The viscous compositions may be in theform of gels, lotions, ointments, creams and the like (e.g., fortransdermal administration) and will typically contain a sufficientamount of a thickening agent so that the viscosity is from about 2500 to6500 cps, although more viscous compositions, even up to 10,000 cps maybe employed. Viscous compositions have a viscosity preferably of 2500 to5000 cps, since above that range they become more difficult toadminister. However, above that range, the compositions may approachsolid or gelatin forms, which are then easily administered as aswallowed pill for oral ingestion.

Liquid preparations are normally easier to prepare than gels, otherviscous compositions, and solid compositions. Additionally, liquidcompositions are somewhat more convenient to administer, especially byinjection or orally. Viscous compositions, on the other hand, may beformulated within the appropriate viscosity range to provide longercontact periods with mucosa, such as the lining of the stomach or nasalmucosa.

Obviously, the choice of suitable carriers and other additives willdepend on the exact route of administration and the nature of theparticular dosage form, e.g., liquid dosage form (e.g., whether thecomposition is to be formulated into a solution, a suspension, gel oranother liquid form), or solid dosage form (e.g., whether thecomposition is to be formulated into a pill, tablet, capsule, caplet,time release form or liquid-filled form).

Solutions, suspensions and gels, normally contain a major amount ofwater (preferably purified water) in addition to the active compound.Minor amounts of other ingredients such as pH adjusters (e.g., a basesuch as NaOH), emulsifiers or dispersing agents, buffering agents,preservatives, wetting agents, jelling agents, (e.g., methylcellulose),colors and/or flavors may also be present. The compositions may beisotonic, i.e., it may have the same osmotic pressure as blood andlacrimal fluid.

The desired isotonicity of the compositions of this invention may beaccomplished using sodium chloride, or other pharmaceutically acceptableagents such as dextrose, boric acid, sodium tartrate, propylene glycolor other inorganic or organic solutes. Sodium chloride is preferredparticularly for buffers containing sodium ions.

Viscosity of the compositions may be maintained at the selected levelusing a pharmaceutically acceptable thickening agent. Methylcellulose ispreferred because it is readily and economically available and is easyto work with. Other suitable thickening agents include, for example,xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer,and the like. The preferred concentration of the thickener will dependupon the agent selected. The important point is to use an amount thatwill achieve the selected viscosity. Viscous compositions are normallyprepared from solutions by the addition of such thickening agents.

A pharmaceutically acceptable preservative may be employed to increasethe shelf-life of the compositions. Benzyl alcohol may be suitable,although a variety of preservatives including, for example, parabens,thimerosal, chlorobutanol, or benzalkonium chloride may also beemployed. A suitable concentration of the preservative will be from0.02% to 2% based on the total weight although there may be appreciablevariation depending upon the agent selected.

Those skilled in the art will recognize that the components of thecompositions should be selected to be chemically inert with respect tothe active compound. This will present no problem to those skilled inchemical and pharmaceutical principles, or problems may be readilyavoided by reference to standard texts or by simple experiments (notinvolving undue experimentation), from this disclosure and the documentscited herein.

It is generally envisaged that compounds and compositions of theinvention will be administered by injection, as such compounds are toelicit anti-HIV antibodies, and the skilled artisan may, from thisdisclosure and the knowledge in the art, formulate compounds andcompositions identified by herein methods for administration byinjection and administer such compounds and compositions by injection.

The inventive compositions of this invention are prepared by mixing theingredients following generally accepted procedures. For example theselected components may be simply mixed in a blender, or other standarddevice to produce a concentrated mixture which may then be adjusted tothe final concentration and viscosity by the addition of water orthickening agent and possibly a buffer to control pH or an additionalsolute to control tonicity. Generally the pH may be from about 3 to 7.5.Compositions may be administered in dosages and by techniques well knownto those skilled in the medical arts taking into consideration suchfactors as the age, sex, weight, and condition of the particularpatient, and the composition form used for administration (e.g., solidvs. liquid). Dosages for humans or other mammals may be determinedwithout undue experimentation by the skilled artisan, from thisdisclosure, the documents cited herein, and the knowledge in the art.

Suitable regimes for initial administration and further doses or forsequential administrations also are variable, may include an initialadministration followed by subsequent administrations; but nonetheless,may be ascertained by the skilled artisan, from this disclosure, thedocuments cited herein, and the knowledge in the art.

The present invention will be further illustrated in the followingExamples which are given for illustration purposes only and are notintended to limit the invention in any way.

EXAMPLES Example 1: HIV Vaccine Design to Target Germline Precursors ofGlycan-Dependent Broadly Neutralizing Antibodies

See Steichen et al., Immunity Volume 45, Issue 3, 20 Sep. 2016, Pages483-496, the disclosure of which is incorporated by reference.

Broadly neutralizing antibodies (bnAbs) against the N332 supersite ofthe HIV Envelope (Env) trimer are the most common bnAbs induced duringinfection, making them promising leads for vaccine design. Wild-type Envglycoproteins lack detectable affinity for supersite-bnAb germlineprecursors and are therefore unsuitable immunogens to primesupersite-bnAb responses. Applicants employed mammalian cell surfacedisplay to design stabilized Env trimers with affinity forgermline-reverted precursors of PGT121-class supersite bnAbs. Thetrimers maintained native-like antigenicity and structure, activatedPGT121 inferred-germline B cells ex vivo when multimerized on liposomes,and primed PGT121-like responses in PGT121 inferred-germline knock-inmice. Design intermediates have levels of epitope modification betweenwild-type and germline-targeting trimers; their mutation gradientsuggests sequential immunization to induce bnAbs, in which thegermline-targeting prime is followed by progressively less-mutateddesign intermediates and lastly with native trimers. The vaccine designstrategies described could be utilized to target other epitopes on HIVor other pathogens.

A vaccine is needed for global HIV prevention. Broadly neutralizingantibodies (bnAbs) directed against relatively conserved epitopes in theotherwise highly antigenically variable HIV Envelope (Env) glycoproteintrimer offer important guides for vaccine design. BnAbs have beenisolated from a small minority of HIV-infected individuals and have beenshown to protect against challenge in various animal models, but havenot been induced by vaccination in humans or standard animal models(Burton and Hangartner, 2016; Mascola and Haynes, 2013; West et al.,2014). BnAbs recovered from natural infection are typically highlymutated (Klein et al., 2013a; Mouquet et al., 2010; Pancera et al.,2010; Scheid et al., 2009; Walker et al., 2011; Xiao et al., 2009; Zhouet al., 2010) and many also contain insertions and/or deletions (Kepleret al., 2014), owing to chronic stimulation of B cells by mutating Env.Many bnAbs also possess unusually long or short heavy chaincomplementarity determining region 3 loops (Scheid et al., 2011; Walkeret al., 2011; Walker et al., 2009; Wu et al., 2011; Zhou et al., 2010),and some are polyreactive (Haynes et al., 2005). Less mutated bnAbs withfewer unusual features have been engineered, offering more tractablegoals for consistent vaccine elicitation (Georgiev et al., 2014; Jardineet al., 2016b; Sok et al., 2013). Overall, bnAb elicitation byvaccination presents a major challenge.

Recombinant native-like trimers are promising HIV vaccine componentsbecause they contain the conformational epitopes of most known bnAbs andlack many non-neutralizing epitopes present on less native constructs(Julien et al., 2013; Kong et al., 2016; Kwon et al., 2015; Lyumkis etal., 2013; Pancera et al., 2014; Sanders et al., 2013; Scharf et al.,2015). However, native-like trimers have features that may impede bnAbinduction: they are highly glycosylated and expose both strain-specificneutralizing epitopes and non-neutralizing epitopes. Immunization withnative-like trimers in standard mouse, rabbit and macaque models hasthus far elicited either non-neutralizing antibodies (Hu et al., 2015)or neutralizing antibodies only against the immunogen strain (de Taeyeet al., 2016; Sanders et al., 2015) analogous to the strain-specificresponses to the seasonal flu vaccine in humans. Induction of HIV bnAbswill likely require development of vaccination strategies that focusresponses to relatively conserved, sub-dominant epitopes and avoid orsuppress responses to non-neutralizing and strain-specific epitopes.

Germline targeting, a vaccine priming strategy to initiate the affinitymaturation of select germline-precursor B cells, could help solve thisimmunofocusing problem by preferentially activating bnAb precursors(Dimitrov, 2010; Xiao et al., 2009). The strategy aims to activatebnAb-precursor B cells, induce productive (bnAb-like) somatic mutations,and produce memory B cells that can be boosted subsequently to selectadditional productive mutations (Dosenovic et al., 2015; Jardine et al.,2015). For some bnAbs, inferred precursors have affinity for Env fromparticular HIV isolates (Andrabi et al., 2015; Doria-Rose et al., 2014;Gorman et al., 2016; Liao et al., 2013), facilitating design of primingimmunogens based on Env from those isolates (Haynes et al., 2012). Forother bnAbs, efforts to identify wild-type Env that bind inferredprecursors have failed (Hoot et al., 2013; Jardine et al., 2013; McGuireet al., 2013; Scheid et al., 2011; Xiao et al., 2009; Zhou et al.,2010). These latter cases require design of modified Env to serve as apriming immunogen (Dimitrov, 2010; Pancera et al., 2010; Xiao et al.,2009; Zhou et al., 2010). Proof of principle that designedgermline-targeting immunogens can activate their intended precursors andgenerate a potentially boostable memory response was recentlydemonstrated in knock-in mice with B cell precursors for VRCO1-classbnAbs directed to the CD4-binding site (Dosenovic et al., 2015; Jardineet al., 2015; McGuire et al., 2016). Following a germline-targetingprime, induction of bnAbs is expected to require a succession of boosts,driving a succession of germinal center reactions, in order to selectsufficient mutations (Dimitrov, 2010; Dosenovic et al., 2015; Haynes etal., 2012; Jardine et al., 2013; 2015; 2016b; Klein et al., 2013b; Liaoet al., 2013; McGuire et al., 2013; 2016; Pancera et al., 2010; Wu etal., 2011; Xiao et al., 2009; Zhou et al., 2010). Supporting the conceptthat sequential immunization with different immunogens will be requiredto develop a bnAb response, native-like trimers but notgermline-targeting immunogens were found to boost near-bnAb B cells(bearing a mature VRC01-class bnAb heavy chain) to induce bnAbs(Dosenovic et al., 2015).

Glycan-dependent bnAbs in general, and N332-supersite bnAbs inparticular, are important targets for germline-targeting vaccine design.In a recent longitudinal study of HIV infection in Africa, more thanhalf of the HIV-infected individuals who produced bnAb responsesproduced them against glycan-directed epitopes, the majority of whichwere within the N332 supersite (Landais et al., 2016). The prevalence ofN332-supersite bnAb responses is probably due in part to the highaccessibility of their epitopes on top of the trimer.

Among N332-supersite bnAbs, PGT121-class bnAbs have been particularlywell characterized, providing strong rationale for germline-targetingefforts. PGT121-class bnAbs are among the most potent bnAbs (Mouquet etal., 2012; Walker et al., 2011), and PGT121 delivered passively tomacaques protects against SHIV infection (Moldt et al., 2012; Shingai etal., 2014) and can suppress viremia when delivered after infection(Barouch et al., 2013; Shingai et al., 2013). However, PGT121-classinferred precursors show no measureable affinity for wild-type Envproteins that have been evaluated (Mouquet et al., 2012; Sok et al.,2013). Thus, development of a priming immunogen for PGT121-classprecursors requires either design of a modified Env or identification ofa natural Env with PGT121-class germline-binding capacity. Crystalstructures have been determined for several PGT121-class bnAbs incomplex with either BG505 SOSIP native-like trimers or gp120 (Garces etal., 2015; Garces et al., 2014; Julien et al., 2013; Kong et al., 2016;Pancera et al., 2014), and for unliganded structures of twogermline-reverted PGT121 variants (Mouquet et al., 2012; Sok et al.,2013), providing critical information to guide design of modified Envfor PGT121-class germline-targeting.

PGT121-class bnAbs interact with conformationally flexible structures onHIV Env, including several glycans and the V1 variable loop, makingcomputational design of germline-targeting Env challenging. Here,Applicants developed a structure-guided directed evolution approach,using mammalian cell surface display, to design PGT121-classgermline-targeting stabilized-trimer immunogens. Applicants multimerizedthese trimers on liposomes, and evaluated trimer and liposome immunogensby biophysical, structural, and ex vivo B cell activation analyses.Applicants further evaluated germline-targeting trimers by vaccinationin PGT121 inferred-germline knock-in mice. Applicants' design processproduced design intermediates with increasing levels of epitopemodification between wild-type and germline-targeting trimers. Theseresults led to Applicants' hypothesizing prime-boosting strategies inwhich a germline-targeting prime is followed by boosts withprogressively less-modified design intermediates and then with wild-typeEnv followed ultimately by a cocktail of Env variants to expand breadth.Evaluation of several of these prime-boosting strategies in PGT121germline and chimeric knock-in mice is described in a related study(Escolano et al., 2016).

Design of Germline-Targeting Gp120s.

Applicants identified mammalian cell surface display as a desirableplatform for engineering modified HIV Env constructs with affinity forinferred-germline PGT121 Abs, as it should allow for optimization ofmonomeric or multimeric antigens bearing mammalian glycans (Chen et al.,2008). Therefore, Applicants developed a lentivirus-based mammalian cellsurface display method to carry out directed evolution of HIV gp120monomers and gp140 trimers (FIG. 9). Structural analysis of the PGT121interaction with gp120 (Julien et al., 2013; Pancera et al., 2014) ledus to hypothesize that the V1 and V3 loops were the key sites forgermline-targeting mutations. For selection agents, Applicants assembleda collection of six germline-reverted Abs, all using heavy chain genesVH4-59, D3-3 and J6 and light chain genes V3-21 and J3, with varyingdegrees of mutation in the D gene and L-CDR3 and with differences in thenon-templated regions at the V-D and D-J boundaries (FIG. 10). Ranked bysimilarity to the germline D and L3 sequences, these Abs are:GL_(CDR3rev1) (most similar to germline D/L3), GL_(CDR3rev2),GL_(CDR3rev3), GL_(CDR3rev4), GL_(CDR3rev5), and GL_(CDR3mat) (germlineV and J genes but mature CDR3 loops). Applicants began by screeninglibraries based on BG505 T332N gp120 and BG505 SOSIP T332N gp140(Sanders et al., 2013) in which the conserved glycosylation site atposition 332 absent in BG505 was introduced (FIG. 6A). These moleculeshad no detectable affinity for germline-reverted PGT121 Abs (FIG. 6B andTable 1). Therefore, Applicants employed a “boot-strapping” approach:for initial screening Applicants utilized two variants of GL_(CDR3mat),one with nine PGT121 light chain mutations (GL+9, with 28 μM affinityfor BG505 T332N gp120) and another with three light chain mutations(GL+3, no detectable affinity for BG505 T332N gp120) (FIG. 10).Screening a gp120 random mutagenesis library for binding to GL+9 led tothe molecule 3MUT, with mutations T135A and T139I to eliminate the V1loop glycosylation sites at positions 133 and 137 (FIG. 6A). Screening agp140 structure-guided V1 loop library for binding to GL+3 led to theisolation of 5MUT, with four different mutations (V134Y, N136P, I138L,D140N) in the V1 loop. Combining the mutations in 3MUT and 5MUT produced7MUT gp120, Applicants' first construct with quantifiable affinity forGL_(CDR3mat) (K_(D)=44 μM, FIGS. 6A and 6B). To improve this affinityfurther, Applicants screened a gp120 V1 and V3 loop saturationmutagenesis library for binding to GL_(CDR3mat); combining the mostenriched mutations (N137F, T320F, Q328M) with 7MUT produced 10MUT, withK_(D)≈1 μM for GL_(CDR3mat) (FIGS. 6A and 6B). Finally, to increaseaffinity and breadth, Applicants screened a gp120 V1 loop directedmutagenesis library for binding to GL_(CDR3rev2) and GL_(CDR3rev4) (FIG.10). This approach culminated in 11MUT_(B), with K_(D)s of ˜5 μM, ˜3 μMand ˜8 μM for GL_(CDR3rev1), GL_(CDR3rev3) and GL_(CDR3rev5),respectively, and detectable but not quantifiable binding toGL_(CDR3rev4) (FIGS. 6A, 11, and Table 1). Thus, mammaliandisplay-directed evolution enabled the design of germline-targetinggp120 molecules with appreciable affinity for PGT121 germline-revertedantibodies.

Design of Stabilized and Germline-Targeting Trimers.

For initial design of germline-targeting and boosting trimers,Applicants transferred the germline-targeting mutations from the gp120versions of 3MUT, 5MUT, 7MUT and 10MUT onto the BG505 SOSIP trimerplatform. These molecules displayed characteristics of native-liketrimers, such as high affinity for the trimer-specific bnAb PGT151(Falkowska et al., 2014) and a melting temperature (T_(m)) similar toBG505 SOSIP (FIG. 7A). Furthermore, all had similar monovalentaffinities for PGT121 and GL_(CDR3mat) as their gp120 counterparts (FIG.7A), indicating that the germline-targeting mutations were transferableto a native-like trimer.

In addition to binding bnAb putative precursors, germline-targetingtrimers should have an otherwise native-like antigenic profile, withhigh affinity for bnAbs and no significant affinity for non-neutralizingantibodies directed to epitopes exposed on monomeric gp120 but buried orconformationally absent on the trimer. BG505 SOSIP gp140, the trimer onwhich Applicants' PGT121-class germline-targeting designs were based,displays undesirable binding to V3 non-neutralizing antibodies (Sanderset al., 2013) (FIGS. 7B and 12) and induces non-neutralizing V3responses in mice, rabbits, and macaques (de Taeye et al., 2016; Hu etal., 2015; Sanders et al., 2015), indicating that this trimer samplesconformational states that expose non-neutralizing epitopes.Furthermore, BG505 SOSIP gp140 displayed on mammalian cells via a PDGFRlinker showed strong binding to trimer-structure-dependent bnAbs (PGT151and PGT145) (Falkowska et al., 2014; Walker et al., 2011) but also tonon-neutralizing antibodies directed to the V3 loop (4025) (Gorny etal., 2011) and the CD4-binding site (b6) (Barbas et al., 1992) (notshown), suggesting the coexistence on the cell surface of native-liketrimers along with non-native trimers, dimers and/or monomers.Applicants also found that adding germline-targeting mutations to BG505SOSIP reduced the already-modest expression by 50% (FIG. 7A). Therefore,Applicants sought to use mammalian display-directed evolution to improvethe antigenic profile, thermal stability and expression level of theBG505 SOSIP trimer and germline-targeting trimers.

Applicants' trimer improvement effort focused on two types of libraries:(i) whole gene saturation mutagenesis libraries and (ii) a combinatoriallibrary sampling the one or two most common HIV residues at Envpositions where BG505 uses rare (frequency <10%) HIV residues (FIG. 7C).The “rare” library, which allowed variation at eleven positions in gp120and two in gp41, was screened for binding to trimer structure-preferringbnAbs PGT145, PGT151 and PG16 and for lack of binding tonon-neutralizing antibodies b6 and 4025. This yielded the Rare3 clonewith five mutations in gp120 (T106E, M271I, F288L, T290A, N363Q) andwith the expression level improved by a factor of ˜2 and the meltingtemperature (T_(M)) increased by 1.4° C. (FIG. 13). The saturationmutagenesis library was constructed in three segments, two coveringgp120 and one for gp41 (FIG. 7C). Next generation sequencing andbioinformatics were employed to analyze the results of the first twosorts (Jardine et al., 2016a), while Sanger sequencing was used toidentify enriched clones that survived four or five sorts. Enrichedmutations from both sequencing methods were combined and tested insoluble trimers and were also assembled into combinatorial libraries andre-screened using the same antibodies as before. The gp41 libraryproduced MD2, with an L568D point mutation that increased expressionlevels by a factor of ˜4, and MD33, with four additional mutations(F519S, A561P, V570H, R585H) that increased the T_(M) by 4° C. andimproved expression by a factor of ˜7 relative to BG505 SOSIP (FIG. 13).The gp120 library produced MD16, with three mutations (F223W, R304V,A319Y) and reduced binding to V3 non-neutralizing antibodies (FIG. 12).Finally, mutations from Rare3, MD16 and MD33 were combined to produceMD39 with 11 mutations (F223W and T290A did not improve the biophysicalproperties of the trimer and were excluded; data not shown). Compared toBG505 SOSIP.D664, the MD39 yield improved by a factor of ˜7, T_(M)increased by ° C., and antigenic profile improved by reduced V3 Abreactivity and similar bnAb binding except for slightly reducedaffinities for V2 apex bnAbs (FIGS. 7A, 7B and 13)

Combining the MD39 mutations with germline-targeting mutations producedgermline-targeting trimers with improved properties. MD39-10MUT had6-fold improved yield and 6° C. higher T_(M) compared to 10MUT (FIGS. 7Aand 13). Applicants' most advanced germline-targeting trimer,MD39-11MUT_(B), the only trimer with detectable affinity for five of sixPGT121-germline reverted variants tested (FIG. 7B), had excellent yield,thermal stability and antigenic profile (FIG. 7A, B). Directed evolutiontherefore produced native-like trimers with improved potentialfunctionality via both stabilization and germline-targeting mutations.

Structural Analysis.

To ascertain whether stabilized, PGT121-germline-targeting trimersmaintain native-like structure, Applicants conducted crystallography andelectron microscopy (EM) studies. Negative stain EM two-dimensionalclassification revealed that all four trimers tested (MD39, 10MUT,MD39-10MUT, and MD39-11MUT_(B)) were characterized by a high fraction(≥95%) of native-like structural features and were similar in appearanceto BG505 SOSIP. The MD39 mutations improved the structural uniformity ofthe 10MUT trimer, as the amount of flexible, native open conformationsdropped from 35% to 5% between 10MUT and MD39-10MUT (see methods fordescription of 2D classification system). Applicants' bestgermline-targeting trimer, MD39-11MUT_(B), exhibited 100% native closedconformations and was indistinguishable from BG505 SOSIP by EM. Forhigher resolution analysis, Applicants solved a 4.5 Å resolution crystalstructure of MD39-10MUT_(A), a variant of MD39-10MUT with one mutationadded and another removed (Supplemental Methods), complexed with 35022and PGT124 (Garces et al., 2014; Sok et al., 2013). While thisresolution precluded analyses of side-chain conformations, and theinterface between trimer and PGT124 could not be analyzed due to missingV1 loop density, the structure accurately determined the backbonepositions for most (1659 of 1692) residues of gp140. Superposition ofthe gp140 backbones in this structure and in the 3.0 Å structure ofBG505 SOSIP N137A complexed with 3H109L and 35022 (PDBid: 5CEZ) or the3.1 Å structure of BG505 SOSIP bound to PGT122 and 35022 (PDBid: 4TVP)gives backbone rmsd values of 0.7 and 1.1 A, respectively. Applicantsconclude that MD39-10MUT_(A), with 20 mutations relative to BG505 SOSIPT332N, retains an overall native-like conformation.

Sequential Boosting Strategies.

As noted above, induction of bnAbs following a germline-targeting primeis expected to require sequential boosting with epitope variants tomature the response. With PGT121-class germline-targeting candidates(10MUT and 11MUT_(B)) in hand, Applicants developed boosting strategiesaiming to select PGT121-like mutations and induce bnAbs. Applicantshypothesized that any sequential immunization strategy starting with agermline-targeting trimer should end with a native-like trimer, such asBG505 MD39 SOSIP, so as to select mutations productive for high-affinityinteraction with the native trimer on the virus. However, in order forPGT121-class antibodies to engage their epitope including the N137glycan on the V1 loop, such antibodies must accommodate V1 loops diversenot only in sequence but also in length and number of glycosylationsites (FIG. 8A-C), implying that boosting with a single native-liketrimer bearing a single V1 loop may not be sufficient. Indeed, boostingonly with a BG505 native-like trimer would present a V1 loop that issignificantly shorter than most (FIG. 8B). Furthermore, modeling ofvariable loops and glycan conformations (not shown) suggested thatdiversity in the V2 and V4 loops might potentially impact the PGT121epitope by altering conformational sampling of the V1 loop or N332glycan, respectively (FIGS. 8A-C), and immunodominant responsesinvolving V2 or V4 could potentially sterically interfere withPGT121-class boosting. Based on these considerations, Applicantshypothesized that a cocktail of native-like trimers displaying diversevariants of the V1, V2, and V4 loops, especially variants within“hotspots” of more frequently-occurring combinations of length andnumber of glycosylation sites (FIG. 8B), might be needed to selectPGT121-class mutations favoring neutralization breadth. Applicantstherefore designed and produced four native-like trimers based on BG505MD39 SOSIP containing diverse loops for V1, V2, V4, and V5 (FIGS. 8C,14A and 14B). These trimers, together with BG505 MD39 SOSIP, form afive-member variable loop cocktail (VLC) that might broaden PGT121-likeresponses initiated by a germline-targeting trimer (FIG. 8D).

Applicants then considered the question of what intermediate boosts, ifany, might be employed between a germline-targeting prime and anative-like trimer. Applicants' germline-targeting design intermediatesbecome increasingly more native-like in the PGT121 epitope (e.g. 7MUT,5MUT and 3MUT have six, four and two epitope mutations, respectively),but the 5MUT and 3MUT mutations are mutually exclusive (3MUT lacks twoV1 glycans while 5MUT has those glycans but has four other V1 loopmutations) (FIG. 6A). These considerations impose directionality on anyboosting scheme (e.g. 7MUT should not be used after 5MUT or 3MUT or WT,and 3MUT should not be used after 5MUT), thus limiting the number ofpossible schemes (Table 2). Considering only the most efficientdirectional schemes, those employing boosting pairs that differ by morethan one mutation or involve substantial affinity changes (Table 3),Applicants identified a total of seven potential boosting schemes (FIG.8E).

Applicants sought to rank these schemes to allow prioritization forexperimental testing. Applicants reasoned that the least mutatedantibody that shows measurable affinity for all of the potentialboosting immunogens, GL+9, could serve as a proxy for intermediatePGT121-class antibodies developing after a germline-targeting prime andbefore a native-like boost. Applicants further reasoned that theaffinity drop, the ratio of GL+9 affinities for two immunogens, could beused to estimate the likelihood of successfully boosting memory B cellswhen the two immunogens are used in sequence (e.g. the GL+9 K_(D)s for7MUT and 3MUT are 3 nM and 1600 nM respectively, so when immunizing with7MUT followed by 3MUT, the affinity drop would be 1600/3=530). Oneexpects that a boost immunogen with very different epitope structurefrom the previous immunogen may result in too large an affinity drop toactivate memory B cells generated by the prior immunogen. Applicantsestimated the affinity drops for all seven boosting schemes (FIG. 8E),ranked them according to the largest affinity drop in that boostingscheme, and listed the three most likely to succeed (FIG. 8F).

In collaborating work, Escolano and colleagues (Escolano et al., 2016)evaluated boosting schemes following the 10MUT trimer prime in PGT121germline (GL_(CDR3-rev4)) knock-in mice and PGT121mature-heavy-chain/germline-light-chain knock-in mice. Relying on thedirectionality of the boosting immunogens developed here, Escolano etal. used serum ELISA against boost candidates after each immunization toselect the most native-like directional boost for which at least weakserum reactivity could be detected; that process resulted in the testingof the second scheme in FIG. 8F and the finding that this scheme inducesPGT121-like bnAbs with substantial breadth and potency. While the firstscheme in 12F remains to be tested, the data in Escolano et al. supportthe validity of the logic underlying these boosting schemes.

Applicants note that the affinity drop analysis also provides clues onhow to improve boosting schemes: to minimize the probability of a boostfailure at a high affinity drop, one could redesign immunogens toequalize the affinity drops in any given scheme. Thus thegermline-targeting design process is capable of defining potential boostimmunogens and directional boosting schemes, and it can guideprioritization and improvement of such schemes.

Germline-targeting vaccine design offers the potential to initiate theinduction of specific classes of protective antibodies against HIV orother pathogens that have eluded vaccine development. Many protectivebnAbs against HIV are directed toward glycan-dependent epitopes on thetrimeric glycoprotein spike (Burton and Hangartner, 2016). Therefore,methods are needed to develop trimer immunogens for germline targetingand boosting of glycan-dependent bnAbs. Trimer immunogens should bestabilized, to maximize the probability of retaining native-likeconformational epitopes in vivo and to minimize the probability ofeliciting non-neutralizing Abs that could potentially detract frompriming or boosting the targeted bnAb responses.

Here, Applicants (1) developed a mammalian cell surface display directedevolution method for optimization of multimeric antigens bearing humanglycans; (2) engineered stabilized HIV Env trimers with affinity forboth germline and mature PGT121-class glycan-dependent bnAbs; (3) showedby crystallography and electron microscopy that these trimers maintainnative-like conformations; (4) demonstrated that germline-targetingtrimers multimerized on liposomes potently activate PGT121 germline andmature B cells ex vivo; and (5) showed that soluble germline-targetingtrimers can prime PGT121-class responses in vivo, in a PGT121inferred-germline knock-in mouse. Applicants' data indicate that11MUT_(B) trimers and trimer-liposomes are promising candidates forpriming PGT121-class glycan-dependent bnAb responses in immune systemswith diverse antibody repertoires, although the frequency ofPGT121-class precursors in humans and the germline-targetingaffinities/avidities necessary to prime those precursors remain to bedetermined.

This work may provide a more general template for HIV bnAbgermline-targeting compared to previous work on germline-targeting forVRCO1-class bnAbs directed to the CD4-binding site. VRCO1-class bnAbsgenerally do not depend on glycans for their activity, evidenced by thefact that elimination of glycans surrounding the VRCO1 epitope generallyincreases neutralization potency (Jardine et al., 2016b); this has ledto removal of all epitope-proximal glycans from germline-targetingcandidates (Jardine et al., 2013; 2015; 2016a; McGuire et al., 2013;2014; 2016). However, the activity of many HIV bnAbs requires engagementof one or more glycans within their epitope, and germline-targetingprimes should probably retain such key glycans, as was the case herewith the N332 glycan. Furthermore, owing to the relative inaccessibilityof the VRCO1 epitope on native-like trimers, efforts to designVRCO1-class germline-targeting primes have converged on strategies toincrease epitope exposure by presentation on minimal domains rather thanon trimers (Jardine et al., 2013; 2015; 2016a; McGuire et al., 2013;2014; 2016), although boosting with native-like trimers is anticipatedto be required to mature the response (Jardine et al., 2016b). Incontrast, many proteo-glycan epitopes are well exposed on native-liketrimers, and some are formed only on intact trimers, making native-liketrimers like those designed here the preferred platform forgermline-targeting. Indeed, multiple different bnAbs could potentiallybe primed with a single trimer harboring multiple germline-targetingepitopes.

As germline-targeting vaccine design requires developing not only thevaccine prime but also boost immunogens to mature the response in orderto elicit bnAbs, Applicants developed both a stabilized native-liketrimer (MD39) and a cocktail of native-like trimers (VLC cocktail) thatcould be employed as boosts to refine and expand the breadth ofresponses initiated by a germline-targeting prime. However, consideringthat memory B cells induced by the germline-targeting prime may not besufficiently mutated to be boosted by a native-like trimer, intermediateboosts may be needed to mature the response prior to native-like boosts.In the process of developing PGT121-class germline-targeting immunogens,Applicants created design intermediates with increasing levels ofepitope modification between wild-type and germline-targeting trimers.These molecules are candidate boost immunogens that if used in sequenceoffer directional and gradual epitope changes to guide maturation of theB cell response. Seven potential sequential immunization schemes wereproposed, and Applicants' analysis of affinity drops provided a rankingof those schemes. In a related paper (Escolano et al., 2016), a subsetof these prime-boosting schemes were evaluated in PGT121 germlineknock-in mice and PGT121 mature-heavy-chain/germline-light-chainknock-in mice, and one such scheme was found to be effective forinducing bnAbs in both mouse models, supporting the germline-targetingvaccine design process described here and encouraging its expanded useand further improvement.

While here Applicants have described strategies for designing trimerimmunogens with changes in the structure of an epitope in order to primeand mature an epitope-specific response, ultimate success of thesestrategies may also require modification of antigenic surfaces outsidethe epitope of interest, to minimize boosting of off-target responsesthat might hinder or interfere with the desired epitope-specificresponse.

The approaches employed here could be adapted for immunogen design toother bnAb targets on HIV and other pathogens. The “bootstrapping”strategy of using partially mutated antibodies (such as GL+3 and GL+9)as initial selection agents and then using antibodies closer to germlinein successive iterations, could be useful for design ofgermline-targeting and boosting immunogens for other bnAbs, such as HIVV2 Apex glycan-dependent bnAbs (Andrabi et al., 2015; Gorman et al.,2016) or influenza virus hemagglutinin stem-directed bnAbs. Applicants'mammalian display methods allowing directed evolution on native-liketrimers should be useful in those endeavors and could also be used tostabilize monomeric or multimeric glycoprotein immunogens for diverseviral vaccines.

In summary, Applicants have developed stabilized native-like trimerimmunogens for germline-targeting and boosting of glycan-dependentPGT121-class bnAb responses against HIV. The immunogens and boostingschemes Applicants created are candidates for human vaccine testing andfurther optimization, while the methods developed here are applicable toimmunogen design for other epitopes and pathogens and thus are ofrelevance for future vaccine design.

DNA Gene Synthesis and Protein Production.

Genes were synthesized by GenScript. Gp120s, gp140s, Fabs and IgGs wereexpressed in 293 cells and purified as described below.

Library Assembly.

The BG505 SOSIP whole gene saturation mutagenesis and “rare amino acid”libraries were synthesized by Integrated DNA Technologies and GenScript,respectively. Libraries for germline targeting were created by errorprone PCR (gene morph II Agilent), site directed mutagenesis (QuikChangeAgilent) or 2-step assembly PCR of degenerate primers using the Q5High-Fidelity DNA Polymerase (New England Biolabs) and cloned into amodified version of the gateway cloning entry vector pENTR/D-TOPO (Otaet al., 2012) using the circularpolymeraseextension cloning (CPEC)method (Quan and Tian, 2014) or Gibson Assembly (New England Biolabs)according to manufacturer's instructions. All libraries were thentransferred to the lentiviral vector pLenti CMVTRE3G puro Dest (Ota etal., 2012) using the LR Clonase II enzyme mix (Thermo Scientific).

Lentivirus Production and Stable Cell Generation.

293T cells cultured in Advanced DMEM (Gibco) supplemented with 5% FCS,GlutaMAX (Gibco), 2-mercaptoethanol (Gibco) and Antibiotic-Antimycotic(Gibco) were co-transfected with 10.8 μg pLenti CMVTRE3G puro Dest genelibrary, 7.0 μg psPAX2 and 3.8 μg pMD2. G as described (Salmon andTrono, 2007). 293T cells stably expressing rtTA3G from the pLenti CMVrtTA3G Blast vector (Ota et al., 2012) were transduced at low moi (<0.1)in a T75 or T225 flask in the presence of 10 μg/mL blasticidin and after24 h transferred to medium supplemented with 2 μg/mL puromycin.

Cell Surface Expression and FACS.

293T cells containing the stable library were induced with doxycycline(1 μg/mL) and harvested the next day in FACS buffer (HBSS, 1 mM EDTA,0.5% BSA). Cells containing BG505-SOSIP libraries were transfected withfurin 24 h prior to induction. Cells were stained with IgGs or Fabs for˜30 min, washed with FACS buffer, and then stained with fluoresceinisothiocyanate (FITC)-labeled α-cMyc (Immunology ConsultantsLaboratory). IgGs were labeled with phycoerythrin (PE)-conjugatedα-human IgG (Sigma), Fabs containing HA epitope tags (PGT145, PGT151,and PG16) were labeled with α-HA-PE (Miltenyi Biotec) and Fabscontaining V5 epitope tags (B6 and 4025) were labeled with α-V5-FITC(GeneTex). Cells were sorted on a BD Influx (BD Biosciences) FACSsorter. Approximately 2×10⁵ double positive cells were collected andexpanded for ˜one week in the presence of puromycin and blasticidinbefore the next round of enrichment. Once the desired population hadbeen obtained, chromosomal DNA was extracted from the cell culture usingthe GenElute Mammalian Genomic DNA Miniprep Kit (Sigma). The gp120 orgp140 gene was PCR amplified from the genomic DNA and inserted back intothe pENTR vector using CPEC cloning or Gibson assembly, transformed intotop10 competent cells (Invitrogen) and colonies were sequenced atGenewiz.

Next Generation Sequencing (NGS).

Sequencing and bioinformatic analysis of the BG505-SOSIP whole genesaturation mutagenesis libraries were done essentially as describedpreviously (Jardine et al., 2016a).

Trimer-conjugated liposome synthesis and characterization. Unilamellarliposomes comprised of DSPC:cholesterol:DGS-NTA(Ni) lipids in a66.5:28.5:5 mole ratio were synthesized by lipid film rehydration andmembrane extrusion, followed by post-synthesis binding of 6×His-taggedtrimer (“6×His” disclosed as SEQ ID NO: 114) for 2 hrs at 4° C.Unconjugated trimer was removed by size exclusion chromatography. Totalconjugated trimer was quantified by ELISA in the presence of 1% triton-Xand 100 mM imidazole to fully disrupt liposomes and Ni-6×Hisinteractions (“6×His” disclosed as SEQ ID NO: 114), respectively, foruninhibited detection via an α-6×His antibody (“6×His” disclosed as SEQID NO: 114). Antigenic profiles were determined by ELISA on intactliposomes.

Ca²⁺-Flux Measurements and Immunizations.

Details about Ca²⁺-flux assays and mouse immunizations can be found inExtended experimental procedures and in (Escolano et al., 2016).

Negative-Stain Electron Microscopy.

Purified SOSIP trimers were analyzed by negative stain EM using aprotocol adapted from (de Taeye et al., 2016).

Differential scanning calorimetry (DSC) and Surface plasmon resonance(SPR) methods are described in the Extended experimental procedures.

TABLE 1 The binding affinities of germline targeting gp120s, related toFIG. 6. BG505- gp120 PGT121 3H3L GL + 9 GL + 3 GL_(CDR3-mat)GL_(CDR3-rev5) GL_(CDR3-rev4) WT (T332N) 7.5 25028000 >128000 >128000 >8000 >84000 2MUT 2.7 — 4900 >40000 — — >400003MUT 4.6 19 1600 >28000 >21000 — >28000 5MUT 5.7 2.5 18 WB WB — >340006MUT 1.4 — 19 >24000 — — >24000 7MUT 1.2 0.25 3 12200 44000 — >360009MUT_(A) 0.57 — — 2700 2900 — >70000 9MUT_(B) 1.5 28 — 29000 WB— >107000 10MUT 0.59 0.04 1.2 1200 790 WB >150000 10MUT-KO 435 — —— >21000 — >21000 11MUT_(A) — — — — 1200 — WB 11MUT_(B) 0.15 0.075 0.6600 840   7700 WB BG505- GL_(Hrev4) 121_(H) gp120 GL_(CDR3-rev3)GL_(CDR3-rev2) GL_(CDR3-rev1) 121_(L) GL_(L-rev4) WT (T332N) >128000— >8000 600 >38000 2MUT — — — 63 >40000 3MUT >11000 — — 22 >28000 5MUT —— — 6 13000 6MUT — — — 5 >24000 7MUT — — — 1.3 >36000 9MUT_(A) — — — —57000 9MUT_(B) — — — 220 39000 10MUT WB >150000 WB — 47000 10MUT-KO — —— 20000 >21000 11MUT_(A) — — — — 51000 11MUT_(B) 3000 —   5200 — —Values are K_(D)s (nM) measured by SPR. WB, weak binding, notquantified. —, not measured.

TABLE 2 Sequential boosting pairs that were eliminated based onviolation of directionality, related to FIG. 8. Sequential boosting pairDirectionality violation 11B → 10/9A 11B contains the native residueN137 which is mutated to F in 10/9A 6 → 3 6 contains the nativeglycosylation site at N133 which is mutated in 3 5 → 3 5 contains nativeglycosylation sites at N133 and N137 and both are mutated in 3 5 → 2 5contains native glycosylation site at N137 which is mutated in 2 Anyboosting pair in which the first immunogen contains a native residuethat is mutated in the second immunogen is a violation ofdirectionality. The immunogen names have “MUT” removed for simplicity.

TABLE 3 Characteristics of sequential boosting paris that obeydirectionality, related to FIG. 8. # of AA Sequential # of AA closerboosting pair Affinity drop^(#) changes to WT comment 11B/10 → 7 Small(5/3) 6/3 4/3 Shown in FIG. 8. 11B/10 → 6 Medium (32/16) 7/4 5/4 Shownin FIG. 8. 11B/10 → 5 Medium (30/15) 7/5 6/5 Shown in FIG. 8. 11B/10 → 3Large (2700/1300) 9/7 8/7 Shown in FIG. 8. 11B/10 → WT Large(47000/23000) 10/9  10/9  Shown in FIG. 8. 7 → 5 Small (6) 2 2 Shown inFIG. 8. 7 → 3 Medium (530) 4 4 Shown in FIG. 8. 7 → WT Large (9300) 6 6Shown in FIG. 8. 6 → WT Large (1500) 5 5 Shown in FIG. 8. 5 → WT Large(1600) 4 4 Shown in FIG. 8. 3 → WT Medium (18) 2 2 Shown in FIG. 8.11B/10 → 2 Large (8200/4100) 10/8  9/8 Would be followed by: 2 → WT 10 →9A Small (4)* 1 1 Small affinity drop and only 1 mutation, thusinefficient 7 → 6 Small (6) 1 1 Small affinity drop and only 1 mutation,thus inefficient 6 → 5 Small (1) 1 1 Small affinity drop and only 1mutation, thus inefficient 6 → 2 Medium (260) 4 4 Would be followed by:2 → WT 3 → 2 Small (3) 1 1 Small affinity drop and only 1 mutation, thusinefficient 2 → WT Small (6) 1 1 Small affinity drop and only 1mutation, thus inefficient ^(#)Affinity drops were calculated based onbinding to the GL + 9 antibody, as described in the text, except wherenoted otherwise. Affinity drops were defined as small (<10), medium(10-1000), or large (>1000). *Affinity drops were calculated based onbinding to the GL_(CDR3-mat) antibody. The immunogen names have “MUT”removed for simplicity. WT, BG505-T332N.

REFERENCES

-   Andrabi, R., Voss, J. E., Liang, C. H., Briney, B., McCoy, L. E.,    Wu, C. Y., Wong, C. H., Poignard, P., and Burton, D. R. (2015).    Identification of Common Features in Prototype Broadly Neutralizing    Antibodies to HIV Envelope V2 Apex to Facilitate Vaccine Design.    Immunity 43, 959-973.-   Barbas, C. F., 3rd, Bjorling, E., Chiodi, F., Dunlop, N., Cababa,    D., Jones, T. M., Zebedee, S. L., Persson, M. A., Nara, P. L.,    Norrby, E., et al. (1992). Recombinant human Fab fragments    neutralize human type 1 immunodeficiency virus in vitro. Proceedings    of the National Academy of Sciences of the United States of America    89, 9339-9343.-   Barouch, D. H., Whitney, J. B., Moldt, B., Klein, F., Oliveira, T.    Y., Liu, J., Stephenson, K. E., Chang, H. W., Shekhar, K., Gupta,    S., et al. (2013). Therapeutic efficacy of potent neutralizing    HIV-1-specific monoclonal antibodies in SHIV-infected rhesus    monkeys. Nature 503, 224-228.-   Burton, D. R., and Hangartner, L. (2016). Broadly Neutralizing    Antibodies to HIV and Their Role in Vaccine Design. Annu Rev Immunol    34, 635-659.-   Chen, K. C., Wu, C. H., Chang, C. Y., Lu, W. C., Tseng, Q.,    Prijovich, Z. M., Schechinger, W., Liaw, Y. C., Leu, Y. L., and    Roffler, S. R. (2008). Directed evolution of a lysosomal enzyme with    enhanced activity at neutral pH by mammalian cell-surface display.    Chem Biol 15, 1277-1286.-   de Taeye, S. W., Moore, J. P., and Sanders, R. W. (2016). HIV-1    Envelope Trimer Design and Immunization Strategies To Induce Broadly    Neutralizing Antibodies. Trends Immunol 37, 221-232.-   Dimitrov, D. S. (2010). Therapeutic antibodies, vaccines and    antibodyomes. mAbs 2, 347-356.-   Doria-Rose, N. A., Schramm, C. A., Gorman, J., Moore, P. L.,    Bhiman, J. N., DeKosky, B. J., Ernandes, M. J., Georgiev, I. S.,    Kim, H. J., Pancera, M., et al. (2014). Developmental pathway for    potent V1V2-directed HIV-neutralizing antibodies. Nature 509, 55-62.-   Dosenovic, P., von Boehmer, L., Escolano, A., Jardine, J.,    Freund, N. T., Gitlin, A. D., McGuire, A. T., Kulp, D. W., Oliveira,    T., Scharf, L., et al. (2015). Immunization for HIV-1 Broadly    Neutralizing Antibodies in Human Ig Knockin Mice. Cell 161,    1505-1515.-   Escolano, A., Steichen, J., Dosenovic, P., Kulp, D. W., Golijanin,    J., Sok, D., Freund, N. T., Gitlin, A. D., Oliveira, T., Araki, T.,    et al. (in-press). Sequential Immunization Elicits Broadly    Neutralizing anti-HIV-1 Antibodies in Ig Knock-in Mice. Cell.-   Falkowska, E., Le, K. M., Ramos, A., Doores, K. J., Lee, J. H.,    Blattner, C., Ramirez, A., Derking, R., van Gils, M. J., Liang, C.    H., et al. (2014). Broadly neutralizing HIV antibodies define a    glycan-dependent epitope on the prefusion conformation of gp41 on    cleaved envelope trimers. Immunity 40, 657-668.-   Garces, F., Lee, J. H., de Val, N., de la Pena, A. T., Kong, L.,    Puchades, C., Hua, Y., Stanfield, R. L., Burton, D. R., Moore, J.    P., et al. (2015). Affinity Maturation of a Potent Family of HIV    Antibodies Is Primarily Focused on Accommodating or Avoiding    Glycans. Immunity 43, 1053-1063.-   Garces, F., Sok, D., Kong, L., McBride, R., Kim, H. J.,    Saye-Francisco, K. F., Julien, J. P., Hua, Y., Cupo, A., Moore, J.    P., et al. (2014). Structural evolution of glycan recognition by a    family of potent HIV antibodies. Cell 159, 69-79.-   Georgiev, I. S., Rudicell, R. S., Saunders, K. O., Shi, W., Kirys,    T., McKee, K., O'Dell, S., Chuang, G. Y., Yang, Z. Y., Ofek, G., et    al. (2014). Antibodies VRC01 and 10E8 neutralize HIV-1 with high    breadth and potency even with Ig-framework regions substantially    reverted to germline. Journal of immunology 192, 1100-1106.-   Gorman, J., Soto, C., Yang, M. M., Davenport, T. M., Guttman, M.,    Bailer, R. T., Chambers, M., Chuang, G. Y., DeKosky, B. J.,    Doria-Rose, N. A., et al. (2016). Structures of HIV-1 Env V1V2 with    broadly neutralizing antibodies reveal commonalities that enable    vaccine design. Nat Struct Mol Biol 23, 81-90.-   Gorny, M. K., Sampson, J., Li, H., Jiang, X., Totrov, M., Wang, X.    H., Williams, C., O'Neal, T., Volsky, B., Li, L., et al. (2011).    Human anti-V3 HIV-1 monoclonal antibodies encoded by the VH5-51/VL    lambda genes define a conserved antigenic structure. PLoS One 6,    e27780.-   Haynes, B. F., Fleming, J., St Clair, E. W., Katinger, H., Stiegler,    G., Kunert, R., Robinson, J., Scearce, R. M., Plonk, K., Staats, H.    F., et al. (2005). Cardiolipin polyspecific autoreactivity in two    broadly neutralizing HIV-1 antibodies. Science 308, 1906-1908.-   Haynes, B. F., Kelsoe, G., Harrison, S. C., and Kepler, T. B.    (2012). B-cell-lineage immunogen design in vaccine development with    HIV-1 as a case study. Nature biotechnology 30, 423-433.-   Hoot, S., McGuire, A. T., Cohen, K. W., Strong, R. K., Hangartner,    L., Klein, F., Diskin, R., Scheid, J. F., Sather, D. N., Burton, D.    R., et al. (2013). Recombinant HIV envelope proteins fail to engage    germline versions of anti-CD4bs bNAbs. PLoS pathogens 9, e1003106.-   Hu, J. K., Crampton, J. C., Cupo, A., Ketas, T., van Gils, M. J.,    Sliepen, K., de Taeye, S. W., Sok, D., Ozorowski, G., Deresa, I., et    al. (2015). Murine Antibody Responses to Cleaved Soluble HIV-1    Envelope Trimers Are Highly Restricted in Specificity. J Virol 89,    10383-10398.-   Jardine, J., Julien, J. P., Menis, S., Ota, T., Kalyuzhniy, O.,    McGuire, A., Sok, D., Huang, P. S., MacPherson, S., Jones, M., et    al. (2013). Rational HIV immunogen design to target specific    germline B cell receptors. Science 340, 711-716.-   Jardine, J., Sok, D., Julien, J. P., Briney, B., Sarkar, A., Adachi,    Y., Dewanji, D., Hsueh, J., Jones, M., Kalyuzhniy, I., et al    (2016b). Minimally Mutated HIV-1 Broadly Neutralizing Antibodies to    Guide Reductionish Vaccine Design. PLoS Pathogens.-   Jardine, J. G., Kulp, D. W., Havenar-Daughton, C., Sarkar, A.,    Briney, B., Sok, D., Sesterhenn, F., Ereno-Orbea, J., Kalyuzhniy,    O., Deresa, I., et al. (2016a). HIV-1 broadly neutralizing antibody    precursor B cells revealed by germline-targeting immunogen. Science    351, 1458-1463.-   Jardine, J. G., Ota, T., Sok, D., Pauthner, M., Kulp, D. W.,    Kalyuzhniy, O., Skog, P. D., Thinnes, T. C., Bhullar, D., Briney,    B., et al. (2015). Priming a broadly neutralizing antibody response    to HIV-1 using a germline-targeting immunogen. Science 349, 156-161.-   Julien, J. P., Cupo, A., Sok, D., Stanfield, R. L., Lyumkis, D.,    Deller, M. C., Klasse, P. J., Burton, D. R., Sanders, R. W.,    Moore, J. P., et al. (2013). Crystal structure of a soluble cleaved    HIV-1 envelope trimer. Science 342, 1477-1483.-   Kepler, T. B., Liao, H. X., Alam, S. M., Bhaskarabhatla, R., Zhang,    R., Yandava, C., Stewart, S., Anasti, K., Kelsoe, G., Parks, R., et    al. (2014). Immunoglobulin gene insertions and deletions in the    affinity maturation of HIV-1 broadly reactive neutralizing    antibodies. Cell Host Microbe 16, 304-313.-   Klein, F., Diskin, R., Scheid, J. F., Gaebler, C., Mouquet, H.,    Georgiev, I. S., Pancera, M., Zhou, T., Incesu, R. B., Fu, B. Z., et    al. (2013a). Somatic mutations of the immunoglobulin framework are    generally required for broad and potent HIV-1 neutralization. Cell    153, 126-138.-   Klein, F., Diskin, R., Scheid, J. F., Gaebler, C., Mouquet, H.,    Georgiev, I. S., Pancera, M., Zhou, T., Incesu, R. B., Fu, B. Z., et    al. (2013a). Somatic mutations of the immunoglobulin framework are    generally required for broad and potent HIV-1 neutralization. Cell    153, 126-138.-   Klein, F., Mouquet, H., Dosenovic, P., Scheid, J. F., Scharf, L.,    and Nussenzweig, M. C. (2013b). Antibodies in HIV-1 vaccine    development and therapy. Science 341, 1199-1204.-   Kong, R., Xu, K., Zhou, T., Acharya, P., Lemmin, T., Liu, K.,    Ozorowski, G., Soto, C., Taft, J. D., Bailer, R. T., et al. (2016).    Fusion peptide of HIV-1 as a site of vulnerability to neutralizing    antibody. Science 352, 828-833.-   Kwon, Y. D., Pancera, M., Acharya, P., Georgiev, I. S., Crooks, E.    T., Gorman, J., Joyce, M. G., Guttman, M., Ma, X., Narpala, S., et    al. (2015). Crystal structure, conformational fixation and    entry-related interactions of mature ligand-free HIV-1 Env. Nat    Struct Mol Biol 22, 522-531.-   Landais, E., Huang, X., Havenar-Daughton, C., Murrell, B., Price, M.    A., Wickramasinghe, L., Ramos, A., Bian, C. B., Simek, M., Allen,    S., et al. (2016). Broadly Neutralizing Antibody Responses in a    Large Longitudinal Sub-Saharan HIV Primary Infection Cohort. PLoS    pathogens 12, e1005369.-   Liao, H. X., Lynch, R., Zhou, T., Gao, F., Alam, S. M., Boyd, S. D.,    Fire, A. Z., Roskin, K. M., Schramm, C. A., Zhang, Z., et al.    (2013). Co-evolution of a broadly neutralizing HIV-1 antibody and    founder virus. Nature 496, 469-476.-   Lyumkis, D., Julien, J. P., de Val, N., Cupo, A., Potter, C. S.,    Klasse, P. J., Burton, D. R., Sanders, R. W., Moore, J. P.,    Carragher, B., et al. (2013). Cryo-EM structure of a fully    glycosylated soluble cleaved HIV-1 envelope trimer. Science 342,    1484-1490.-   Mascola, J. R., and Haynes, B. F. (2013). HIV-1 neutralizing    antibodies: understanding nature's pathways. Immunological reviews    254, 225-244.-   McGuire, A. T., Hoot, S., Dreyer, A. M., Lippy, A., Stuart, A.,    Cohen, K. W., Jardine, J., Menis, S., Scheid, J. F., West, A. P., et    al. (2013). Engineering HIV envelope protein to activate germline B    cell receptors of broadly neutralizing anti-CD4 binding site    antibodies. The Journal of experimental medicine 210, 655-663.-   McGuire, A. T., Gray, M. D., Dosenovic, P., Gitlin, A. D.,    Freund, N. T., Petersen, J., Correnti, C., Johnsen, W., Kegel, R.,    Stuart, A. B., et al. (2016). Specifically modified Env immunogens    activate B-cell precursors of broadly neutralizing HIV-1 antibodies    in transgenic mice. Nat Commun 7, 10618.-   Moldt, B., Rakasz, E. G., Schultz, N., Chan-Hui, P. Y., Swiderek,    K., Weisgrau, K. L., Piaskowski, S. M., Bergman, Z., Watkins, D. I.,    Poignard, P., et al. (2012). Highly potent HIV-specific antibody    neutralization in vitro translates into effective protection against    mucosal SHIV challenge in vivo. Proceedings of the National Academy    of Sciences of the United States of America 109, 18921-18925.-   Mouquet, H., Scharf, L., Euler, Z., Liu, Y., Eden, C., Scheid, J.    F., Halper-Stromberg, A., Gnanapragasam, P. N., Spencer, D. I.,    Seaman, M. S., et al. (2012). Complex-type N-glycan recognition by    potent broadly neutralizing HIV antibodies. Proceedings of the    National Academy of Sciences of the United States of America 109,    E3268-3277.-   Mouquet, H., Scheid, J. F., Zoller, M. J., Krogsgaard, M., Ott, R.    G., Shukair, S., Artyomov, M. N., Pietzsch, J., Connors, M.,    Pereyra, F., et al. (2010). Polyreactivity increases the apparent    affinity of anti-HIV antibodies by heteroligation. Nature 467,    591-595.-   Ota, T., Doyle-Cooper, C., Cooper, A. B., Huber, M., Falkowska, E.,    Doores, K. J., Hangartner, L., Le, K., Sok, D., Jardine, J., et al.    (2012). Anti-HIV B Cell lines as candidate vaccine biosensors.    Journal of immunology 189, 4816-4824.-   Pancera, M., McLellan, J. S., Wu, X., Zhu, J., Changela, A.,    Schmidt, S. D., Yang, Y., Zhou, T., Phogat, S., Mascola, J. R., et    al. (2010). Crystal structure of PG16 and chimeric dissection with    somatically related PG9: structure-function analysis of two    quaternary-specific antibodies that effectively neutralize HIV-1. J    Virol 84, 8098-8110.-   Pancera, M., Zhou, T., Druz, A., Georgiev, I. S., Soto, C., Gorman,    J., Huang, J., Acharya, P., Chuang, G. Y., Ofek, G., et al. (2014).    Structure and immune recognition of trimeric pre-fusion HIV-1 Env.    Nature 514, 455-461.-   Pugach, P., Ozorowski, G., Cupo, A., Ringe, R., Yasmeen, A., de Val,    N., Derking, R., Kim, H. J., Korzun, J., Golabek, M., et al. (2015).    A native-like SOSIP.664 trimer based on an HIV-1 subtype B env gene.    J Virol 89, 3380-3395.-   Quan, J., and Tian, J. (2014). Circular polymerase extension    cloning. Methods in molecular biology 1116, 103-117.-   Salmon, P., and Trono, D. (2007). Production and titration of    lentiviral vectors. Current protocols in human genetics/editorial    board, Jonathan L Haines [et al] Chapter 12, Unit 12 10.-   Sanders, R. W., Derking, R., Cupo, A., Julien, J. P., Yasmeen, A.,    de Val, N., Kim, H. J., Blattner, C., de la Pena, A. T., Korzun, J.,    et al. (2013). A next-generation cleaved, soluble HIV-1 Env trimer,    BG505 SOSIP.664 gp140, expresses multiple epitopes for broadly    neutralizing but not non-neutralizing antibodies. PLoS pathogens 9,    e1003618.-   Sanders, R. W., van Gils, M. J., Derking, R., Sok, D., Ketas, T. J.,    Burger, J. A., Ozorowski, G., Cupo, A., Simonich, C., Goo, L., et    al. (2015). HIV-1 neutralizing antibodies induced by native-like    envelope trimers. Science 349, aac4223.-   Scharf, L., Wang, H., Gao, H., Chen, S., McDowall, A. W., and    Bjorkman, P. J. (2015). Broadly Neutralizing Antibody 8ANC195    Recognizes Closed and Open States of HIV-1 Env. Cell 162, 1379-1390.-   Scheid, J. F., Mouquet, H., Feldhahn, N., Seaman, M. S., Velinzon,    K., Pietzsch, J., Ott, R. G., Anthony, R. M., Zebroski, H., Hurley,    A., et al. (2009). Broad diversity of neutralizing antibodies    isolated from memory B cells in HIV-infected individuals. Nature    458, 636-640.-   Scheid, J. F., Mouquet, H., Ueberheide, B., Diskin, R., Klein, F.,    Oliveira, T. Y., Pietzsch, J., Fenyo, D., Abadir, A., Velinzon, K.,    et al. (2011). Sequence and structural convergence of broad and    potent HIV antibodies that mimic CD4 binding. Science 333,    1633-1637.-   Shingai, M., Donau, O. K., Plishka, R. J., Buckler-White, A.,    Mascola, J. R., Nabel, G. J., Nason, M. C., Montefiori, D., Moldt,    B., Poignard, P., et al. (2014). Passive transfer of modest titers    of potent and broadly neutralizing anti-HIV monoclonal antibodies    block SHIV infection in macaques. The Journal of experimental    medicine 211, 2061-2074.-   Shingai, M., Nishimura, Y., Klein, F., Mouquet, H., Donau, O. K.,    Plishka, R., Buckler-White, A., Seaman, M., Piatak, M., Jr.,    Lifson, J. D., et al. (2013). Antibody-mediated immunotherapy of    macaques chronically infected with SHIV suppresses viraemia. Nature    503, 277-280.-   Sok, D., Laserson, U., Laserson, J., Liu, Y., Vigneault, F.,    Julien, J. P., Briney, B., Ramos, A., Saye, K. F., Le, K., et al.    (2013). The effects of somatic hypermutation on neutralization and    binding in the PGT121 family of broadly neutralizing HIV antibodies.    PLoS pathogens 9, e1003754.-   Walker, L. M., Huber, M., Doores, K. J., Falkowska, E., Pejchal, R.,    Julien, J. P., Wang, S. K., Ramos, A., Chan-Hui, P. Y., Moyle, M.,    et al. (2011). Broad neutralization coverage of HIV by multiple    highly potent antibodies. Nature 477, 466-470.-   Walker, L. M., Phogat, S. K., Chan-Hui, P. Y., Wagner, D., Phung,    P., Goss, J. L., Wrin, T., Simek, M. D., Fling, S., Mitcham, J. L.,    et al. (2009). Broad and potent neutralizing antibodies from an    African donor reveal a new HIV-1 vaccine target. Science 326,    285-289.-   West, A. P., Jr., Scharf, L., Scheid, J. F., Klein, F., Bjorkman, P.    J., and Nussenzweig, M. C. (2014). Structural insights on the role    of antibodies in HIV-1 vaccine and therapy. Cell 156, 633-648.-   Wu, X., Zhou, T., Zhu, J., Zhang, B., Georgiev, I., Wang, C., Chen,    X., Longo, N. S., Louder, M., McKee, K., et al. (2011). Focused    evolution of HIV-1 neutralizing antibodies revealed by structures    and deep sequencing. Science 333, 1593-1602.-   Xiao, X., Chen, W., Feng, Y., Zhu, Z., Prabakaran, P., Wang, Y.,    Zhang, M. Y., Longo, N. S., and Dimitrov, D. S. (2009).    Germline-like predecessors of broadly neutralizing antibodies lack    measurable binding to HIV-1 envelope glycoproteins: implications for    evasion of immune responses and design of vaccine immunogens.    Biochemical and biophysical research communications 390, 404-409.-   Zhou, T., Georgiev, I., Wu, X., Yang, Z. Y., Dai, K., Finzi, A.,    Kwon, Y. D., Scheid, J. F., Shi, W., Xu, L., et al. (2010).    Structural basis for broad and potent neutralization of HIV-1 by    antibody VRC01. Science 329, 811-817.

Example 2: Supplemental Experimental Procedures to Example 1

DNA gene synthesis. Genes were synthesized at Genscript, Inc. Gp120 andgp140 variants in pHLsec contained a C-terminal GTKHHHHHH tag (SEQ IDNO: 115). Genes in pENTR contained a C-terminal cMyc epitope followed bya PDGFR transmembrane domain. IgGs were cloned into pFUSEss and Fabswere in a modified version of pFUSEss (pFABss). DNA was maxi-preppedusing a BenchPro 2100.

Protein Production.

BG505-gp120 and variants based on BG505 contained the L111A mutation formore efficient production of monomer compared to other species(Hoffenberg et al., 2013) and the T332N mutation and were expressed in293F cells grown in 293 Freestyle media (Life Technologies) by transienttransfection with 293Fectin (Invitrogen). Protein was harvested from thesupernatant 96 h post transfection and purified by nickel affinitychromatography on a HIS-TRAP column (GE) followed by HiLoad 16/600Superdex 200 size exclusion chromatography (GE Healthcare). Gp140 SOSIPswere expressed in 293F cells grown in 293 Freestyle media by transienttransfection with either 293Fectin or PEI. The protein was purified fromthe supernatant using a HIS-TRAP column, starting with a wash buffer (20mM Imidazole, 500 mM NaCl, 20 mM Na2HPO4) and mixing with elution buffer(500 mM Imidazole, 500 mM NaCl, 20 mM Na2HPO4) using a linear gradient.The trimer fraction was collected and further purified on an S200Increase 10-300 column (GE) in HBS (10 mM HEPES, 150 mM NaCl). Theoligomeric state of the SOSIP trimers were then confirmed by sizeexclusion chromatography-multi-angle light scattering (SECMALS) usingthe DAWN HELEOS II multi-angle light scattering system with OptilabT-rEX refractometer (Wyatt Technology). The trimers were frozen inthin-walled PCR tubes at 1 mg/ml using liquid nitrogen and stored at−80° C. (Jardine et al., 2015). Fabs and mAbs were produced in 293Fcells as described previously (Jardine et al., 2013). Forcrystallography, SOSIP_MD39_10MUTA was expressed in 293S cells.

ELISA Quantification of SOSIP Expression.

BG505 SOSIP variants were expressed using the Freestyle 293F expressionsystem (Thermo Scientific) according to manufacturer's instructions.After 4 days, supernatants were harvested by centrifugation and storedat 4° C. until analysis. Capture ELISAs were performed essentially asdescribed previously (Schiffner et al., 2016). Briefly, ELISA plateswere coated overnight with trimer specific PGT145 Fab at 4 μg/mL in PBSat 4° C. followed by blocking with 2% w/v bovine serum albumin (BSA) inwashing buffer (PBS+0.05% v/v tween20). SOSIP expression supernatantswere diluted 100× in sample buffer (washing buffer+1% w/v BSA) and foreach variant, a standard curve with known concentration of matchingpurified protein was prepared in sample buffer.

Supernatants and standard curves were added to ELISA plates and detectedwith trimer preferring IgG PGT151 at 10 μg/mL in sample buffer. Sampleswere labeled with horseradish peroxidase coupled Fcg-specific anti-humanIgG (Jackson Immunoresearch), developed and stopped with 1-Step UltraTMB-ELISA substrate (Thermo Scientific) as per manufacturer'sinstructions, and optical densities were read at 450 nm and 570 nm.After background subtraction, data were fit to a “one-site specificbinding with hill slope” curve in graphpad prism, and supernatantconcentrations were extrapolated from standard curves.

Surface Plasmon Resonance (SPR).

Kinetics and affinities of antibody-antigen interactions were measuredon a ProteOn XPR36 (Bio-Rad) using GLC Sensor Chip (Bio-Rad) and1×HBS-EP+pH 7.4 running buffer (20× stock from Teknova, Cat. No H8022)supplemented with BSA at 1 mg/ml. Human Antibody Capture Kit was usedaccording to manufacturer's instructions (Cat. No BR-1008-39 from GE) toimmobilize about 6000 RUs of capture mAb onto each flow cell. In atypical experiment, approximately 300-400 RUs of mAbs were captured ontoeach flow cell and analytes were passed over the flow cell at 50 L/minfor 3 min followed by a 5 min dissociation time. Regeneration wasaccomplished using 3M Magnesium Chloride with 180 seconds contact timeand injected four times per cycle. Raw sensograms were analyzed usingProteOn Manager software (Bio-Rad), including interspot and columndouble referencing, and either Equilibrium fits or Kinetic fits withLangmuir model, or both, were employed when applicable. Analyteconcentrations were measured on a NanoDrop 2000c Spectrophotometer usingAbsorption signal at 280 nm (Jardine et al., 2015). Applicants measuredkinetics and affinity of antibody-Fab-fragment antigen interactions onProteOn XPR36 (Bio-Rad) using HTE Sensor Chip (Bio-Rad) and runningbuffer with 20 mM Sodium Phosphate Dibasic, pH 7.4, 500 mM SodiumChloride, 50 mM Imidazole, supplemented with BSA at 1 mg/ml and Tween 20detergent at 0.05% v/v. Applicants used 0.1 M Nickel sulfate asactivation solution. 0.5 M EDTA was Applicants' regeneration solutionwith 300 seconds contact time and injected two times per cycle (one timeeach for vertical and horizontal orientation). Raw sensograms wereanalyzed using ProteOn Manager software (Bio-Rad), interspot and columndouble referencing, Equilibrium or Kinetic with Langmuir model or bothwhere applicable. Analyte concentrations were measured on NanoDrop 2000cSpectrophotometer using Absorption signal at 280 nm.

Design of PGT121 Germline-Targeting Immunogens.

BG505-gp120 T332N fused to the PDGFR transmembrane domain (TM) wassubjected to random mutagenesis using error prone PCR (gene morph IIAgilent), and the resulting PCR product was gel purified and ligatedinto a modified version of the gateway cloning entry vector pENTR/D-TOPO(Ota et al., 2012) using the circular polymerase extension cloning(CPEC) method (Quan and Tian, 2014). The ligated vector containing theerror prone library was purified using the PCR purification kit (Qiagen)and concentrated. The concentrated library was then transformed intoelectroMAX DH5a-E competent cells (Invitrogen) and grown overnight at37° C. in a 125 mL culture. The plasmid was purified using the BenchPro®2100 (Invitrogen) and the gp120 insert was transferred to the lentiviralvector pLenti CMVTRE3G puro Dest (Ota et al., 2012) using the LR ClonaseII enzyme mix (Invitrogen). The LR clonase reaction was scaled up˜10-fold to increase library size. The LR clonase product was againpurified, concentrated and transformed into electroMAX stbl4 competentcells (Invitrogen) and grown overnight at 30° C. in a 125 mL culture.This plasmid DNA was purified and ready for use in transfection. 293Tcells cultured in Advanced DMEM (Gibco) supplemented with 5% FCS,GlutaMAX (Gibco), 2-mercaptoethanol (Gibco) and Antibiotic-Antimycotic(Gibco) were co-transfected with the BG505-gp120 error prone PCR libraryin pLenti CMVTRE3G puro Dest (10.8 μg), psPAX2 (7.0 μg) and pMD2. G (3.8μg) with fugeneHD in a T75 flask (Salmon and Trono, 2007). The cellswere kept at 37° C. for two days and then the media containing the viruswas collected and spun down at 500 g for 5 min. 293T cells stablyexpressing rtTA3G from the pLenti CMV rtTA3G Blast vector (obtained fromDave Nemazee; (Ota et al., 2012)) were transduced at low moi (<0.1) in aT75 or T225 flask in the presence of 10 μg/mL blasticidin. The next daycells were selected with 2 μg/mL puromycin. 293T cells containing thestable library were induced with doxycycline (1 μg/mL) and the followingday were harvested in FACS buffer (HBSS, 1 mM EDTA, 0.5% BSA). Cellswere stained with either the GL+9 or GL+3 Ab for ˜30 min, washed withFACS buffer, and then stained with fluorescein isothiocyanate(FITC)-labeled α-cMyc (Immunology Consultants Laboratory) andphycoerythrin (PE)-conjugated α-human IgG (Sigma). Cells were sorted ona BD Influx (BD Biosciences) FACS sorter. Approximately 2×10 GL+9positive cells were collected and expanded for ˜one week in the presenceof puromycin and blasticidin before the next round of enrichment wascarried out. There was no enrichment for GL+3 positive cells afterseveral rounds of sorting so only the GL+9 positive cells weresequenced. Once the desired population had been obtained the chromosomalDNA was extracted from the cell culture using the GenElute MammalianGenomic DNA Miniprep Kit (Sigma). The BG505-gp120 gene was PCR amplifiedfrom the genomic DNA and ligated back into the Gateway entry vectorusing CPEC cloning and transformed into top10 competent cells. Later inthe design process Gibson assembly was substituted for CPEC cloning.Colonies were sequenced at Genewiz. The sequences were highly enrichedfor two clones, one containing the N137 glycan knockout by the mutationT139I and the other containing the N133 glycan knockout by the mutationT135A in addition to the T139I mutation. These constructs were called2MUT (T332N, T139I) and 3MUT (T332N, T135A, T139I). Measuring theaffinities of gp120-2MUT and gp120-3MUT against a panel of partiallymutated PGT121 Abs (table S1) indicated that knocking out both glycansgave a larger boost in affinity compared to only the N137 glycan-KO so3MUT was used for further designs.

In parallel to screening the error prone PCR library, a combinatoriallibrary was created based on the structure of PGT122 in complex withBG505 SOSIP (PDB IDs 4NCO and 3J5M). Because the initial SOSIPstructures were low resolution and structures of germline PGT121 showedlight chain conformational changes Applicants elected to do a saturationmutagenesis combinatorial library that would roughly cover the length ofthe V1 loop that could potentially interact with germline PGT121 Abs.The library was generated by PCR amplifying the BG505 SOSIP construct intwo partially overlapping fragments. The C-terminal fragment wasamplified with a primer containing the degenerate codon NNK at fourpositions in the V1 loop (V134, N136, 1138, and D140) as well as adegenerate base encoding N or D at position 137. The two PCR productswere ligated together using a second round of PCR, and this second PCRproduct was inserted into the pENTR vector as described above. Theresulting construct was transferred to the pLenti CMVTRE3G puro Destvector, and lentivirus was produced. Stable cells were stained with theGL+3 Ab and α-cMyc, and double positive cells were sorted. This resultedin a binding population that was sequenced and found to be a singleunique clone containing the mutations V134Y, N136P, I138L, D140N. Thisclone was called 5MUT (T332N, V134Y, N136P, I138L, D140N). Thesemutations were combined with the T139I mutation (6MUT) or theT135A/T139I mutations (7MUT).

Next, a saturation mutagenesis scanning library was created on thegp120-7MUT construct using site directed mutagenesis with the QuikChangekit (Agilent Technologies) with a unique NNK/MNN primer pair for eachposition that was scanned. 11 positions in the V1 loop (T132 to M142)and 10 positions in the V3 loop (T320 to Q328) were scanned and theresulting 21 reactions were pooled, purified, concentrated, andtransformed into electroMAX DH5a-E competent cells and transferred topLenti CMVTRE3G puro Dest as described above. This library was thenstained separately with GL_(CDR3mat), GL_(CDR3rev4), or a Chimeric Abcontaining the mature PGT121 heavy chain paired with the GL_(CDR3rev4)light chain (121H/GLL-rev4), as well as α-cMyc for expression. Doublepositive cells were sorted and 3 mutations were enriched in theGL_(CDR3mat) sort (N137F, T320F, Q328M) and two mutations were enrichedin the 121H/GLL-rev4 sort (N135R, Q328M) whereas a binding populationwas not obtained in the GL_(CDR3rev4) sort. Combining these mutationswith 7MUT resulted in 9MUT_(A) (7MUT+N137F/Q328M),9MUT_(B)(7MUT+N135R/Q328M), and 10MUT (7MUT+N137F/T320F/Q328M). The9MUT_(B) protein showed improved binding to 121H/GLL-rev4 but worsebinding to all other PGT121-class antibodies tested compared to 7MUT(from which 9MUT_(B) was derived) and so 9MUT_(B) was not selected forfurther use except as a control for the chimeric antibody (data notshown). Gp120-10MUT showed better binding to GL_(CDR3mat) compared togp120-9MUT_(A) and T320F was used in subsequent designs with theexception of Applicants' SOSIP-10MUT_(A) crystal structure, which lacksthe T320F mutation.

Having established ˜1 μM binding to the GL_(CDR3mat) Ab with 10MUTApplicants' goal was to improve the immunogen to tolerate more variationwithin the H-CDR3. For this Applicants created three more V1 loopcombinatorial libraries each containing four NNK codons. The threelibraries contained NNK codons at positions (A135/P136/F137/L138),(F137/L138/I139/N140), and (I139/N140/D141/M150). Each library wasassembled from two partially overlapping ultramers (Integrated DNATechnologies) and ligated into the gp120-10MUT gene using gibsonassembly (New England Biolabs). The three libraries were pooled andscreened against zup GL_(CDR3rev2) and GL_(CDR3rev4) Abs. Sortingagainst the GL_(CDR3rev4) Ab resulted in enrichment for the D141Nmutation (11MUT_(A)) and sorting against the GL_(CDR3rev2) resulted inenrichment for L139 and S140 with the most frequent clone containing thesequence N137/L138/L139/S140. When these mutations were combined withthe D141N mutation as well as a T415V mutation, which Applicants hadidentified as being beneficial for binding to PGT121 on an engineeredouter domain construct (data not shown), it resulted in 11MUT_(B)Development of BG505-SOSIP_MD39.

BG505 SOSIP “Rare Amino Acid” Library.

The BG505 SOSIP “rare amino acid” library was synthesized at GenScript.It was first sorted against PG16 followed by a sort for a high PGT145/B6binding ratio. The cells were expanded for 1 week and then sorted foreither high PGT145/B6 or high PGT151/4025. After six rounds of sortingthe library was sequenced (Genewiz). PGT145, PGT151, and PG16 Fabscontained HA epitope tags and were labeled with α-HA-PE (MiltenyiBiotec). B6 and 4025 Fabs contained V5 epitope tags and were labeledwith α-V5-FITC (GeneTex).

BG505 SOSIP Whole Gene Saturation Mutagenesis.

The whole gene saturation mutagenesis library was synthesized atIntegrated DNA Technologies in four segments that each contained ˜150NNK codons that were cloned into the BG505-SOSIP gene using either CPECor Gibson assembly which resulted in four libraries. NNK codons werebarcoded with a silent mutation on each side. The libraries created fromthe second and third segments were combined into one. The first, secondand third libraries had NNK codons covering residues Y39-N185, N186-R500and K502-Q658, respectively. The library that covered gp41 (502-658) wassorted for high PGT145/cMyc, high PGT145/B6, and high PGT151/cMyc. Thefirst gp120 library (39-185) was sorted for high PGT145/B6, and highPGT151/4025. The second gp120 library (186-500) was sorted for highPGT145/cMyc, high PGT145/B6, high PGT151/4025, and high PGT151/cMyc. Thesorted libraries were sequenced and analyzed essentially as describedpreviously (Jardine et al., 2016). Positions that enriched for the sameamino acid against multiple different mAb sorts (E.g. PGT145(+)/B6(−)and PGT151(+)/4025(−)) were favored for testing in follow upcombinatorial libraries or directly testing in recombinantly purifiedprotein. Combinatorial libraries based on the next generation sequencinganalysis were assembled from overlapping ultramers and sorted againstthe same antibodies described above.

Differential Scanning Calorimetry (DSC).

DSC experiments were performed on a MicroCal VP-Capillary differentialscanning calorimeter (Malvern Instruments). The HEPES buffered saline(HBS) buffer was used for baseline scans and the protein samples werediluted into HBS buffer to adjust to 0.25 mg/ml. The system was allowedto equilibrate at 20° C. for 15 min and then heat up till 90° C. at ascan rate of 90° C./h. Buffer correction, normalization, and baselinesubtraction were applied during data analysis using Origin 7.0 software.The non-two-state model was used for data fitting.

ELISA to Characterize Antigenic Profile of Native-Like Trimers.

96-well plates were coated overnight at 4° C. with 6×-His Epitope TagAntibody (“6×His” disclosed as SEQ ID NO: 114) (Thermofisher) at 2 mg/mlin PBS. Plates were washed 3 times with PBS, 0.05% Tween (PBS-T), andblocked with 10% milk PBS for 1 h. Subsequently, 2 mg/ml of the purifiedHis-tagged SOSIP protein was added for 2 h in 1% milk PBS-T, after whichthe plates were washed three times with PBS-T. Serial dilutions of mAbsin 1% milk PBS-T were added to the plates for 1 h, after which theplates were washed again three times with PBS-T before the addition ofanti-human Fc region-conjugated alkaline phosphatase (JacksonImmunoresearch) at 1:1000 for 1 h. After four final washes, binding wasdetected by the addition of alkaline phosphatase substrate and measuredby absorbance at 405 nm.

Development of Variable Loop Cocktail (VLC) Trimers.

Using BG505 SOSIP MD39 trimer as a base, a series of new trimers wereengineered by replacing the immunodominant variable loops of the BG505strain with loops from alternative strains. Given the vast number of HIVstrains available, Applicants created three separate criteria to guideloop selection. For the first set of variable loop transplants,Applicants cataloged the number of glycans within each variable loop andthe length of each variable loop (FIG. 7C). Certain combinations ofvariable loop lengths and glycans were observed more frequently thanothers across HIV strains (e.g. 20.48% of HIV strains have a 14 aminoacid variable loop 2 with one glycan, FIG. 7C). Applicants searched forstrains that contain the most common loop length/glycan combination foreach of the variable loops (V1, V2, V4, V5). For the second set ofvariable loop transplants, Applicants searched for strains with variableloops of the same length and number of glycans as BG505, but with verydifferent amino acid sequence and glycan positioning within the loops ascompared to BG505. No single strain had all the same variable looplengths and number of glycans as BG505, so Applicants relaxed criteriaand matched each variable loop independently for this set of variableloop transplants only. For the third set of variable loop transplants,Applicants searched for strains with exceptionally long variable loops(V1, V2, V4 must be ≥4 amino acids longer than BG505). Under each ofthese criteria, Applicants were able to obtain one or two trimers thathad a reasonable level of expression and formed well-behaved native-liketrimers (FIG. 14A). The loops of the VLCs are defined as: VLC-1 (V1:BES10.EF363127, V2: BL8157. DQ886035, V4: BF1P51.JQ250880, V5:CZM197.DQ388515), VLC-2(BG505. DQ208458), VLC-3 (PRLS08.FJ469757)VLC-4(GHJ193.AB231897), VLC-5(OUR2478P.EF165541). A region defined as335-351 (HxB2) underneath V4 was included when transplanting V4, due tohigh variability and close contact with V4. Including BG505, Applicantsreport a set of 5 trimers with diverse variable loops. One version ofthe VLC trimers that did not have the MD39 mutations, and insteadcontained an extra stabilizing disulfide (DS21: V120C-Q315C) in order tostaple down the tip of the V3, this version of the VLC trimers was usedin an accompanying manuscript (Escolano et al., 2016).

The invention is further described by the following numbered paragraphs:

1. A non-naturally occurring protein comprising an immunogen comprisinga gp120 or gp140 trimer comprising one or more stabilizing mutations,cleavage-independent modifications, and/or an anchored membrane.

2. The non-naturally occurring protein of numbered paragraph 1comprising:

(a) a Type I or Type II immunogen, wherein the Type I immunogen is agp120 and the Type II immunogen is a gp140 trimer molecules with one ormore mutations that improve binding to germline-reverted and/orless-mutated versions of PGT121 or

(b) a Type III immunogen, wherein the Type III immunogen is a gp140trimer molecule with one or more stabilizing mutations to increaseexpression level and/or increase thermal melting temperature and/orimprove antigenic profile, where a favorable antigenic profile meansbetter affinity for broadly neutralizing antibodies and no or very weakaffinity for non-neutralizing antibodies or

(c) a Type IV immunogen, wherein the Type IV immunogen is a combinationof one or more mutations from the Type II immunogen and Type IIIimmunogen, wherein the Type IV immunogen is a gp140 trimer comprisingboth stabilizing mutations and germline-targeting mutations or

(d) a Type V immunogen, wherein the Type V immunogen is a trimer with amodified surface or of a strain other than BG505 or

(e) a Type VI immunogen, wherein the Type VI immunogen comprises one ormore additional trimer modifications that add extra functionality andthat can be combined with Types II, III, IV or V or

(f) a Type VII immunogen, wherein the Type VIII immunogen is anative-like trimer from other HIV strains stabilized by MD39 and Olio6mutations or

(g) a Type VIII immunogen, wherein the Type VIII immunogen is acleavage-independent trimer which is a variant of BG505 MD39 that doesnot require cleavage by furin or

(h) a Type IX immunogen, wherein the Type IX immunogen is a glycanmasked trimer in which N-linked glycosylation sites cover the bottom andsides of the soluble trimer or

(i) a Type X immunogen, wherein the Type X immunogen is a native-liketrimer with variable loops V1, V2b and V4 modified to minimize theirlengths and maximize the number of glycosylation sites contained withinthem or

(j) a Type XI immunogen, wherein the Type XI immunogen is a BG505MD39-based, single-component, self-assembling nanoparticle or

(k) a Type XII immunogen, wherein the Type XII immunogen is a BG505MD39-based, membrane-bound native like trimer.

3. The non-naturally occurring protein of numbered paragraph 1 or 2,wherein the protein comprises any one of:

(a) BG505-gp120-L111A-2_T135A_T139I_mC (BG505-gp120 3mut) (SEQ ID NO: 1)VWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISAWDQSLKPCVKLTPLCVTLQCTNVANNIIDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEP(b) BG505-gp120-L111A-2_5mut_mC (BG505-gp120 5mut) (SEQ ID NO: 3)VWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISAWDQSLKPCVKLTPLCVTLQCTNYTPNLTNDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEP(c) BG505-gp120-L111A-2_7mut_mC (BG505-gp120 7mut or BG505 gp120 7MUT)(SEQ ID NO: 5) VWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISAWDQSLKPCVKLTPLCVTLQCTNYAPNLINDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEP(d) BG505-gp120-L111A-2_10mut_mC(BG505-gp120 10mut or BG505 gp120 10MUT) (SEQ ID NO: 7)VWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISAWDQSLKPCVKLTPLCVTLQCTNYAPFLINDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYAFGDIIGDIRMAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEP(e) BG505-gp120-L111A-2_10mut2A_mC (SEQ ID NO: 9)VWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISAWDQSLKPCVKLTPLCVTLQCTNYAPFLINNMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYAFGDIIGDIRMAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEP(f) BG505-gp120-L111A-2_11mut2A_mC (SEQ ID NO: 11)VWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISAWDQSLKPCVKLTPLCVTLQCTNYAPNLLSNMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYAFGDIIGDIRMAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSIVLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEP

4. The non-naturally occurring protein of numbered paragraph 1 or 2r,wherein the trimer comprises any one of:

(a) BG505_SOSIP.D664_JS_3mut_mC (SOSIP-3MUT) (SEQ ID NO: 13)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVANNIIDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (b) BG505_SOSIP.D664_JS_5mut_mC(SOSIP-5MUT) (SEQ ID NO: 15)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNYTPNLTNDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (c) BG505_SOSIP.D664_JS_7mut_mC(SOSIP-7MUT) (SEQ ID NO: 17)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNYAPNLINDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (d) BG505_SOSIP.D664_JS_10mut_mC(SOSIP-10MUT) (SEQ ID NO: 19)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNYAPFLINDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYAFGDIIGDIRMAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (e) BG505_SOSIP.D664_JS_10mut2A_mC(SEQ ID NO: 21) AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNYAPFLINNMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYAFGDIIGDIRMAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (f) BG505_SOSIP.D664_JS_11mut2A_mC(SEQ ID NO: 23) AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNYAPNLLSNMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYAFGDIIGDIRMAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSIVLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD

5. The non-naturally occurring protein of numbered paragraph 1 or 2,wherein the trimer comprises any one of:

(a) BG505_SOSIP_D664_MD39_mC (BG505 SOSIP-MD39 or MD39) (SEQ ID NO: 25)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (b)BG505_SOSIP_D664_MD16_mC stabilizes the V3 loop (SEQ ID NO: 27)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGWAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKDTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLA LD (c)BG505_SOSIP_D664_split1_1_mC increases melting temp 10 C. (SEQ ID NO:29) AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHECVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIIELWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (d)BG505_SOSIP_D664_MD39C_mC highest melting temp 82.5 C. (SEQ ID NO: 31)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHECVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIIELWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKLTVWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (e)BG505_SOSIP_D664_MD9_mC improved yield/stability (SEQ ID NO: 33)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPERQQHLLKDTVWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (f)BG505_SOSIP_D664_MD2_mC improved yield (SEQ ID NO: 35)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKDTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (g)BG505_SOSIP_D664_olio6_mC (SEQ ID NO: 37)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFWRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTAPNNFTVKSIRIGPGQAFYYMGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGMFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKLIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (h)BG505_SOSIP_D664_MD53_mC (SEQ ID NO: 39)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFWRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVLSTQLLLNGSLAEEEVIVRSENITNNAKNILVQLNTPVQINCTAPNNFTVKSIRIGPGQAFYYMGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGMFFFCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKLIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDVWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLA LD (i)BG505_SOSIP_D664_MD37 (SEQ ID NO: 164)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPC C KLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSACTQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNI L VQLNTPVQINCTRPNNNTRKSIRIGPGCAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFA Q SSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQCMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAV S LGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQEHLHKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD

6. A The non-naturally occurring protein of numbered paragraph 1 or 2,wherein the trimer comprises any one of:

(a) BG505_SOSIP_D664_MD39_9mut2A_mC (MD39 + 9MUT) (SEQ ID NO: 42)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNYAPFLINNMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRMAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (b)BG505_SOSIP_D664_MD39_10mut_mC (SEQ ID NO: 44)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNYAPFLINDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYFGDIIGDIRMAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (c)BG505_SOSIP_D664_MD39_10mut2A_mC (SEQ ID NO: 46)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNYAPFLINNMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYFGDIIGDIRMAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (d)BG505_SOSIP_D664_MD39_11mut2A_mC (SEQ ID NO: 48)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNYAPNLLSNMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYFGDIIGDIRMAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSIVLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD

7. The non-naturally occurring protein of numbered paragraph 1 or 2,that can be employed in strategic boosting regimens, wherein the trimercomprises any one of:

(a) BG505_SOSIP_MD39_VLC1-03  (SEQ ID NO: 50)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQ EIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTDWDNAT LANMTGEIKNCSFNMTTELRDKKQKVYSLFYELDIIPIENEYISNNNTSNTSYRLINC NTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVS TQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFY YTGDIIGDIRQAHCNVSATQWEQTLKGIAAKLLEHFGNNTIIRFAQSSGGDLEVTTHS FNCGGEFFYCNTSGLFNGSSWNLNKTKENTTNLENGTITLPCRIKQIINMWQRIGQA MYAPPIQGVIRCVSNITGLILTRDGGNKSAGIETFRPGGGDMRDNWRSELYKYKVVKI EPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLI CCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDL  LALD (b) BG505_SOSIP_MD39_VLC2-04  (SEQ ID NO: 52)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQ EIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCSDYEGNTT RQNITMKEEKGEIKNCSFNMTTELRDKKQKVYSLFYKLDITPIEEDNNSNNSSSANS SNSNANYTNYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNN NTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSGTKWKNTLKQIVKKLGDHFGNNTIIRF AQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWNRTNGTWNDVEGLNYTNGNDTI TLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGNDTDKNETFRPGGG DMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAG STMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQQEKNEQDLLALD  (c) BG505_SOSIP_MD39_VLC2-08  (SEQ ID NO: 54)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQ EIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCVTLKNCSN SNCSISRNISIEMDGEIKNCSFNMTTELRDKKQKVYSLFYRLDIVPIESSNNSQLSNNS QVSNNSQSSNYSQYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGT GPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCT RPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKKDWEKTLQQVATKLGQHFGN NTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWIRNSSNSTWNSSASNSTEL NSNITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGHETENKTETFR PGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFL GAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQAR VLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEI SNYTQIIYGLLEESQNQQEKNEQDLLALD  (d) BG505_SOSIP_MD39_VLC3-13 (SEQ ID NO: 56) AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQ EIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNAALTNV TITNGPNITEEIRNCSFNMTTELRDKKQKVYSLFYKLDLVQINGSGGEYRLINCNTSA ITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLL LNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDI IGDIRQAHCNVSGTKWNETLKQVAGKLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCG GEFFYCNTSGLFNSTWPENGTMEGSNGTITLPCRIKQIINMWQRIGQAMYAPPIQGVI RCVSNITGLILTRDGGNSTTDTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRC KRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLL RAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (e) BG50_5SOSIP_VLC1-03_D521_mC  (SEQ ID NO : 58)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQ EIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCCKLTPLCVTLQCTDWDNATL ANMTGEIKNCSFNMTTELRDKKQKVYSLFYELDIIPIENEYISNNNTSNTSYRLINCNTS AITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQL LLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGCAFYATG DIIGDIRQAHCNVSATQWEQTLKGIAAKLLEHFGNNTIIRFANSSGGDLEVTTHSFNCG GEFFYCNTSGLFNGSSWNLNKTKENTTNLENGTITLPCRIKQIINMWQRIGQAMYAPPI QGVIRCVSNITGLILTRDGGNKSAGIETFRPGGGDMRDNWRSELYKYKVVKIEPLGVA PTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQ SNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (f) BG505_SOSIP_VLC2-04_DS21_mC  (SEQ ID NO: 60)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQ EIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCCKLTPLCVTLQCSDYEGNTT RQNITMKEEKGEIKNCSFNMTTELRDKKQKVYSLFYKLDITPIEEDNNSNNSSSANSSN SNANYTNYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVS TVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNN TRKSIRIGPGCAFYATGDIIGDIRQAHCNVSGTKWKNTLKQIVKKLGDHFGNNTIIRFA NSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWNRTNGTWNDVEGLNYTNGNDTITL PCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGNDTDKNETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYL RDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYG LLEESQNQQEKNEQDLLALD  (g) BG505_SOSIP_VLC2-08_DS21_mC  (SEQ ID NO: 62)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQ EIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCCKLTPLCVTLQCVTLKNCSN SNCSISRNISIEMDGEIKNCSFNMTTELRDKKQKVYSLFYRLDIVPIESSNNSQLSNNSQ VSNNSQSSNYSQYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTR PNNNTRKSIRIGPGCAFYATGDIIGDIRQAHCNVSKKDWEKTLQQVATKLGQHFGNNT IIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWIRNSSNSTWNSSASNSTELNSN ITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGHETENKTETFRPGG GDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVFLGFLGAA GSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLA VERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNY TQIIYGLLEESQNQQEKNEQDLLALD  (h) BG505_SOSIP_VLC3-13_DS21_mC (SEQ ID NO: 64) AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQ EIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCCKLTPLCVTLQCTNAALTNV TITNGPNITEEIRNCSFNMTTELRDKKQKVYSLFYKLDLVQINGSGGEYRLINCNTSAIT QACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLN GSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGCAFYATGDII GDIRQAHCNVSGTKWNETLKQVAGKLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGG EFFYCNTSGLFNSTWPENGTMEGSNGTITLPCRIKQIINMWQRIGQAMYAPPIQGVIRC VSNITGLILTRDGGNSTTDTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKR RVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRA PEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (i) AC10_SOSIP_olio6_mC  (SEQ ID NO: 66)AVEQTWVTVYYGVPVWKEANTTLFCASDAKAYNTEVHNVWATHACVPTDPNPQEVELENVTENFNMWKNNMVDQMHEDIISLWDQSLKPCVKLTPLCVTLSCTDNVGN DTSTNNSRWDKMEKGEIKNCSFNITTNMRDKMQKQYALFWKLDVVPIEEGKNNNSSF TDYRLISCNTSVITQACPKVTFEPIPIHYCAPAGFALLKCKDKKFNGTGPCKNVSTVQC THGIKPVVSTQLLLNGSLAEEEVVIRSENFSNNARTIIVQLNTSVEIKCIAPNNFTVKGIH IGPGRAFYYMGDIIGDIRQAHCNISRQNWNNTLKQIAEKLREQFGNKTIVFRQSSGGDPEIVMHTFNCAGMFFYCNTAELFNSTWYANGTISIGGGNKTNIILPCRIKLFINMWQEVG KAMYAPPISGQIRCSSNITGLLLTRDGGRGNQTDNQTEIFRPVGGDMKNNWRSELYKY KVVRIEPLGIAPTRCKRRVVGRRRRRRAVGIGALSLGFLGAAGSTMGAASMTLTVQA RLLLSGIVQQQNNLLRAPEPQQHLLQDTHWGIKQLQARVLAVEHYLRDQQLLGIWGC SGKLICCTAVPWNVSWNNRSVDDIWENMTWMQWDREISNYTSLIYTLIEESQNQQEK  NEQELLALD (j) BG505_SOSIP_SET224_4_mC(BG505_SOSIP_R4_mC)  (SEQ ID NO: 68)AENLWVTVYYGVPVWKDAETTLFCASDAKAYSTEKHNVWATHACVPTDPNPQ EVVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLNCTELNDTN TTATNSSGRVIEDKEIKNCSFNMTTSLRDKVQRVYSLFNKFDIVPIDNSNDSYRLISCN TSAITQACPKVSFEPIPIHYCAPAGFAILKCNDKEFNGTGPCKSVSTVQCTHGIRPVVST QLLLNGSLAEEEVIIRSENFTNNAKTILVQLNEPVVINCTRPNNNTVKSIRIGPGQAFYY TGEIIGDIRQAHCTVSRETWNKTLGRVVEQLREQFRNKTIIVFNQSSGGDPEIVMHSF NCGGEFFYCNSTQLFNSTWYGNETETGGTNDTIGNITLPCRIKQIINMWQEVGKAMY APPIRGQISCSSNITGLILTRDGGNNNETNTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKCKRRVVGRRRRRRAVGIGAMSLGFLGAAGSTMGAASLTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLI CCTNVPWNTSWSNKSLDQIWDNMTWLEWDREISNYTQLIYNLLEESQNQQEKNEQD  LLALD 

8. The non-naturally occurring protein of numbered paragraph 1 or 2,wherein the trimer comprises any one of:

(a) BG505_SOSIP_MD39_congly_mC (SEQ ID NO: 70)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (b)BG505_SOSIP_MD39_CtCys_mC (SEQ ID NO: 72)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLAL DGTKHHHHHHC(c) BG505_SOSIP_MD39_CD4KO4_mC (SEQ ID NO: 74)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGTDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (d)BG505_SOSIP_D664_olio6_CD4KO4_mC (SEQ ID NO: 76)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFWRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTAPNNFTVKSIRIGPGQAFYYMGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGMFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKLIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGTDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (e)AC10_SOSIP_olio6_congly_mC (SEQ ID NO: 78)AVEQTWVTVYYGVPVWKEANTTLFCASDAKAYNTEVHNVWATHACVPTDPNPQEVELENVTENFNMWKNNMVDQMHEDIISLWDQSLKPCVKLTPLCVTLSCTDNVGNDTSTNNSRWDKMEKGEIKNCSFNITTNMRDKMQKQYALFWKLDVVPIEEGKNNNSSFTDYRLISCNTSVITQACPKVTFEPIPIHYCAPAGFALLKCKDKKFNGTGPCKNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVVIRSENFSNNARTIIVQLNTSVEINCTAPNNFTVKGIHIGPGRAFYYMGDIIGDIRQAHCNISRQNWNNTLKQIAEKLREQFGNKTIVFRQSSGGDPEIVMHTFNCAGMFFYCNTSELFNSTWYANGTISIGGGNKTNIILPCRIKLFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGRGNQTDNQTEIFRPVGGDMKNNWRSELYKYKVVRIEPLGIAPTRCKRRVVGRRRRRRAVGIGALSLGFLGAAGSTMGAASMTLTVQARLLLSGIVQQQNNLLRAPEPQQHLLQDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTAVPWNVSWNNRSVDDIWENMTWMQWDREISNYTSLIYTLIEESQNQQE KNEQELLALD

9. The non-naturally occurring protein of numbered paragraph 1 or 2,wherein the trimer comprises any one of:

(a) 191084_SOSIP_MD39 (SEQ ID NO: 80)TENLWVTVYYGVPVWRDAETTLFCASDAKAYDTEMHNVWATHACVPTDPNPQEIDLENVTEKFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLNCTAITNDTRGNETGINRTVETTEMTNCSFNMTTELRDRKKKVNALFYKLDIVQIGENSSSQYRLINCNTSVITQACPKVTFEPIPIHYCAPAGFAILKCKDKEFNGTGTCRNVSSVQCTHGIKPVVSTQLLLNGSLAEGQVIIRSENISDNAKTIIVQLNESVPINCTRPNNNTVRGIHLGPGQTFFYTDIIGDIRQAHCNVSESKWNKALQEVVKQLRQHWNKTIIFKSSSGGDLEITTHSFNCGGEFFYCNTSGLFNSTWNIAGNRTNDTKSNETITLPCRIKQIVNVWQRVGQAIYAPPIAGVIRCNSNITGLLLVRDGGATNNTDETFRPGGGNMRDNWRSELYKYKVVKIEPLGVAPTRCRRRVVERRRRRRAVGLGAVSIGFLGAAGSTMGAASVTLTVQARQLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLKDQQLLGIWGCSGKLICCTTVPWNSSWSNKSQNEIWDNMTWLQWDKEISNYTQLIYSLIEESQNQQEKNEQELLALD (b)001428_SOSIP_MD39 (SEQ ID NO: 81)VENLWVTVYYGVPVWKEARTTLFCASDAKAYETEVHNVWATHACVPTDPNPQEMVLGNVTENFNMWKNDMVDQMHEDVISLWAQSLKPCVKLTPLCVTLECTQVNATQGNTTQVNVTQVNGDEMKNCSFNTTTEIRDKKQKAYALFYRLDLVPLERENRGDSNSASKYILINCNTSAITQACPKVNFDPIPIHYCTPAGYAILKCNNKTFNGTGSCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIIIRSENLTDNVKTIIVHLDQSVEIVCTRPNNNTVKSIRIGPGQTFYYTGDIIGNIREAHCNISEKKWHEMLRRVSEKLAEHFPNKTIKFTSSSGGDLEITTHSFNCRGEFFYCNTSGLFNSTYMPNGTYMPNGTNNSNSTIILPCRIKQIINMWQEVGRAMYAPPIAGNITCNSNITGLLLVRDGGKNNNTEIFRPGGGDMRDNWRSELYKYKVVEIKPLGVAPTRCKRRVVGRRRRRRAVGLGAVSLGFLGAAGSTMGAASITLTVQARQLLSGIVQQQSNLLQAPEPQQHLLQDTHWGIKQLQTRVLAIEHYLKDQQLLGIWGCSGKLICCTAVPWNSSWSNKSLTDIWDNMTWMQWDREVSNYTGIIYRLLEDSQNQQERNEQ DLLALD (c)AC10_SOSIP_MD39 (SEQ ID NO: 82)AVEQTWVTVYYGVPVWKEANTTLFCASDAKAYNTEVHNVWATHACVPTDPNPQEVELENVTENFNMWKNNMVDQMHEDIISLWDQSLKPCVKLTPLCVTLSCTDNVGNDTSTNNSRWDKMEKGEIKNCSFNITTNMRDKMQKQYALFYKLDVVPIEEGKNNNSSFTDYRLISCNTSVITQACPKVTFEPIPIHYCAPAGFALLKCKDKKFNGTGPCKNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVVIRSENFSNNARTIIVQLNTSVEIKCIRPNNNTVKGIHIGPGRAFYYTGDIIGDIRQAHCNISRQNWNNTLKQIAEKLREQFGNKTIVFRQSSGGDPEIVMHTFNCAGEFFYCNTAELFNSTWYANGTISIGGGNKTNIILPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGRGNQTDNQTEIFRPVGGDMKNNWRSELYKYKVVRIEPLGIAPTRCKRRVVGRRRRRRAVGIGALSLGFLGAAGSTMGAASMTLTVQARLLLSGIVQQQNNLLRAPEPQQHLLQDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTAVPWNVSWNNRSVDDIWENMTWMQWDREISNYTSLIYTLIEESQNQQEK NEQELLALD (d)AC10_SOSIP_olio6 (SEQ ID NO: 83)AVEQTWVTVYYGVPVWKEANTTLFCASDAKAYNTEVHNVWATHACVPTDPNPQEVELENVTENFNMWKNNMVDQMHEDIISLWDQSLKPCVKLTPLCVTLSCTDNVGNDTSTNNSRWDKMEKGEIKNCSFNITTNMRDKMQKQYALFWKLDVVPIEEGKNNNSSFTDYRLISCNTSVITQACPKVTFEPIPIHYCAPAGFALLKCKDKKFNGTGPCKNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVVIRSENFSNNARTIIVQLNTSVEIKCIAPNNFTVKGIHIGPGRAFYYMGDIIGDIRQAHCNISRQNWNNTLKQIAEKLREQFGNKTIVFRQSSGGDPEIVMHTFNCAGMFFYCNTAELFNSTWYANGTISIGGGNKTNIILPCRIKLFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGRGNQTDNQTEIFRPVGGDMKNNWRSELYKYKVVRIEPLGIAPTRCKRRVVGRRRRRRAVGIGALSLGFLGAAGSTMGAASMTLTVQARLLLSGIVQQQNNLLRAPEPQQHLLQDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTAVPWNVSWNNRSVDDIWENMTWMQWDREISNYTSLIYTLIEESQNQQEK NEQELLALD (e)ZM197M_MD39 (SEQ ID NO: 84)MEQLWVTVYYGVPVWKEAKATLFCASDAKAYEKEVHNVWATHACVPTDPNPQEIPLGNVTENFNMWKNDMADQMHEDIISLWDQSLKPCVKLTPLCVTLNCSDATSNTTKNATNTNTTSTDNRNATSNDTEMKGEIKNCTFNITTEVRDRKTKQRALFYKLDVVPLEEEKNSSSKNSSYKEYRLISCNTSTITQACPKVSFDPIPIHYCAPAGYAILKCNNKTFNGTGPCHNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIIIRSENLTDNTKTIIVHLNESVEINCTRPNNNTVKSVRIGPGQTFFYTGEIIGDIRQAHCNLSKSNWTTTLKRIEKKLKEHFNNATIKFESSAGGDLETTTHSFNCRGEFFYCNTSGLFNSSLLNDTDGTSNSTSNATITLPCRIKQIINMWQEVGRAMYASPIAGIITCKSNITGLLLTRDGGNKSAGIETFRPGGGNMKDNWRSELYKYKVVEIKPLGIAPTSCKRRVVERRRRRRAAGIGAVSLGFLGAAGSTMGAASVMLTVQARQLLSGIVQQQSNLLRAPEPQQHMLQDTHWGIKQLQTRVLAIEHYLKDQQLLGLWGCSGKLICCTAVPWNTSWSNKSKDEIWDNMTWMQWDREIDNYTQVIYQLLEVSQNQQEKNENDLLALD (f) B41_SOSIP_D664_MD39 (SEQ ID NO: 85)AAKKWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEIVLGNVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLNCNNVNTNNTNNSTNATISDWEKMETGEMKNCSFNVTTSIRDKIKKEYALFYKLDVVPLENKNNINNTNITNYRLINCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNSKTFNGSGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEIVIRSENITDNAKTIIVQLNEAVEINCTRPNNNTVKSIHIGPGRAFYYTGDIIGNIRQAHCNISKARWNETLGQIVAKLEEQFPNKTIIFNHSSGGDPEIVTHSFNCGGEFFYCNTTPLFNSTWNNTRTDDYPTGGEQNITLQCRIKQIINMWQGVGKAMYAPPIRGQIRCSSNITGLLLTRDGGRDQNGTETFRPGGGNMRDNWRSELYKYKVVKIEPLGIAPTACKRRVVQRRRRRRAVGLGAFSLGFLGAAGSTMGAASMALTVQARLLLSGIVQQQNNLLRAPEPQQHMLQDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKIICCTNVPWNDSWSNKTINEIWDNMTWMQWEKEIDNYTQHIYTLLEVSQIQQEKN EQELLELD

10. The non-naturally occurring protein of numbered paragraph 1 or 2,wherein the trimer comprises any one of:

(a) BG505_SOSIP_MD39_link14 (SEQ ID NO: 86)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGSHSGSGGSGSGGHAAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEK NEQDLLALD (b)BG505_SOSIP_MD39_CP1.1 (SEQ ID NO: 87) VSLGFLGAAGS TMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDT HWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDGGSGSGGGSGSGGSSAENL WVTVYYGVPVWK DAET T LFCAS D AKAYETEKHN V WATHACVPTDPNPQEIHLENVT EEFNMWKN NMVEQMHEDIISLWDQSLKPC V KLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAP A GFAILKCKD K KFNGTGPCPSVSTVQCTH G IKPVVSTQLLLNGS LAEEEV IIRSENITN N AKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTG D IIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCK RRVVG (c)BG505_SOSIP_MD39_CP1.2 (SEQ ID NO: 88) VSLGFLGAAGS TMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDT HWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLGGSGSGGGSGSGGSSGSGLWVT VYYGVPVWK DAETTLFCAS D AKAYETEKHN V WATHACVPTDPNPQEIHLENVTEEF NMWKN NMVEQMHEDIISLWDQSLKPC V KLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPK VSFEPIPIHYCAPA GFAILKCKD K KFNGTGPCPSVSTVQCTH G IKPVVSTQLLLNGSLAE EE V IIRSENITN NAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTG D IIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRR (d)BG505_SOSIP_MD39_CP2 (SEQ ID NO: 89) VSLGFLGAAGS TMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDT HWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTGGSGVTVYYGVPVWK D AET T LFCAS DAKAYETEKHN V WATHACVPTDPNPQEIHLENVTEEFNMWKN N MVEQMHE DIISLWDQSLKPC VKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAP A GFA ILKCKD KKFNGTGPCPSVSTVQCTH G IKPVVSTQLLLNGSLAEEE V IIRSENITN N AKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTG D IIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRGGSGSGVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD (e) BG505_SOSIP_MD39_CP3(SEQ ID NO: 90) VSLGFLGAAGS T MGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDT HWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESGGSGSGAGGLWVTVYYGVPVWK D AET T LFCAS D AKAYETEKHNV WATHACVPTDPNPQEIHLENVTEEFNMWKN N MVEQMHEDIISLWD QSLKPC VKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAP A GFAILKCKD KKFNGTGPCPSVSTVQCTH G IKPVVSTQLLLNGSLAEEE V IIRSENITN N AKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTG D IIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGGGSGSGQNQQEKNEQDLL ALD

11. The non-naturally occurring protein of numbered paragraph 1 or 2,wherein the trimer comprises any one of:

(a) BG505_MD39_GRSF4 (SEQ ID NO: 91)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNSSEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNNQSLLALDNGS (b) BG505_MD39_GRSF7(SEQ ID NO: 92) AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNSSEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNANLSEIWDNMTWLQWDKNISNYTQIIYGLLEESQNQQEKNNQSLLALDNGS (c)BG505_MD39_CP1.2_GRSF4 (SEQ ID NO: 93)VSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLGGNGSGGGSGSGGNGSSGLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNSSEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRR (d) BG505_MD39_link14_GRSF4(SEQ ID NO: 94) AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNSSEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGSHSGSGGSGSGGHAAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNNQSLLALDNGS (e)BG505_MD39_link14_GRSF7 (SEQ ID NO: 95)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNSSEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGSHSGSGGSGSGGHAAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNANLSEIWDNMTWLQWDKNISNYTQIIYGLLEESQNQQEKNNQSLLALDNGS

12. The non-naturally occurring protein of numbered paragraph 1 or 2,wherein the trimer comprises any one of:

(a) BG505_MD39_CP1.2_GRSF4_qLoops1 (SEQ ID NO: 96)VSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLGGNGSGGGSGSGGNGSSGLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNSSEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDNMTGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINGSGGEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWPENGTMEGSNGTITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYK VVKIEPLGVAPTRCKRR(b) BG505_MD39_CP1.2_GRSF7_qLoops1 (SEQ ID NO: 97) GG NSSGSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQ N ESNEQDLGGNGSGGGSGSGGNGSSGLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNSSEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDNMTGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINGSGGEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWPENGTMEGSNGTITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSE LYKYKVVKIEPLGVAPTRC NRS (c) BG505_MD39_GRSF4_qLoops1 (SEQ ID NO: 98)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNSSEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDNMTGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINGSGGEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWPENGTMEGSNGTITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNNQSLLALDNGS

13. The non-naturally occurring protein of numbered paragraph 1 or 2,wherein the trimer comprises any one of:

(a) BG505_SOSIP_MD39_2JD6 (SEQ ID NO: 99)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDGSGGLSERMLKALNDQLNRELYSAYLYFAMAAYFEDLGLEGFANWMKAQAEEEIGHALRFYNYIYDKNGRVELDEIPKPPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKDYSTRAFLEWFINEQVEEEASVKKILDKLKFAKDSPQILFMLDKELSARAP KLPGLLMQGGE**(b) BG505 SOSIP MD39 E2p (also referred to as “MD39-1b5s”)(SEQ ID NO: 100) AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDASGAAAKPATTEGEFPETREKMSGIRRAIAKAMVHSKHTAPHVTLMDEADVTKLVAHRKKFKAIAAEKGIKLTFLPYVVKALVSALREYPVLNT[C/A/T]IDDETEEIIQKHYYNIGIAADTDRGLLVPVIKHADRKPIFALAQEINELAEKARDGKLTPGEMKGASCTITNIGSAGGQWFTPVINHPEVAILGIGRIAEKPIVRDGEIVAAPMLALSLSFDHRMIDGATAQKALNHIKRLLSDPELLLM**

14. The non-naturally occurring protein of numbered paragraph 1 or 2,wherein the trimer comprises any one of:

(a) BG505_MD39_gp160_dCT (SEQ ID NO: 101)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDKWASLWNWFDISNWLWYIKIFIMIVGGLIGLRIVFAVLSVIHRVRQGYSPLS** (b) BG505_MD39_gp160_dCT_GRSF4.1(SEQ ID NO: 102) AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNSSEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDKWASLWNWFDISNWLWYIKIFIMIVGGLIGLRIVFAVLSVIHRVRQGYSPLS** (c) BG505_MD39_gp160_GRSF4.1_m(SEQ ID NO: 103) AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNSSEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDKWASLWNWFDISNWLWYIKIFIMIVGGLIGLRIVFAVLSVIHRVRQGYSPLSFQTHTPNPRGLDRPERIEEEDGEQDRGRSTRLVSGFLALAWDDLRSLCLFCYHRLRDFILIAARIVELLGHSSLKGLRLGWEGLKYLWNLLAYWGRELKISAINLFDTIAIAVAEWTDRVIEIGQRLCRAFLHIPRRIRQGLERALL** (d) BG505_MD39_gp140-PDGFR(SEQ ID NO: 104) AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDGGGSGGSGGSEQKLISEEDLGGSGGSGGSNAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIIS LIILIMLWQKKPR**(e) BG505_MD39_gp140-PDGFR_GRSF4.1_m (SEQ ID NO: 105)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNSSEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDGGGSGGSGGSEQKLISEEDLGGSGGSGGSNAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIIS LIILIMLWQKKPR**(f) BG505_MD39_gp140-PDGFR_link14 (SEQ ID NO: 106)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGSHSGSGGSGSGGHAAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDGGGSGGSGGSEQKLISEEDLGGSGGSGGSNAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR** (g) BG505_MD39_gp160-dCT_link14 (SEQ ID NO: 107)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTPVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGSHSGSGGSGSGGHAAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDKWASLWNWFDISNWLWYIKIFIMIVGGLIGLRIVFAVLSVIHRVRQGYSPLS**(h) BG505_MD39_gp160-dCT_link14_GRSF4.1 (SEQ ID NO: 108)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNSSEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGSHSGSGGSGSGGHAAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDKWASLWNWFDISNWLWYIKIFIMIVGGLIGLRIVFAVLSVIHRVRQGYSPLS**(i) BG505_MD39_gp140-PDGFR_link14_GRSF4.1 (SEQ ID NO: 109)AENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNSSEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGSHSGSGGSGSGGHAAVGIGAVSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDGGGSGGSGGSEQKLISEEDLGGSGGSGGSNAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR** (j) BG505_MD39_gp140-PDGFR_CP1.2_GRSF4.0(SEQ ID NO: 110) VSLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKDTHWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLGG N GSGG GSGSGG NGSSGLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNSSEIHLENVTEEFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCQNVSTVQCTHGIKPVVSTQLLLNGSLAEEEVIIRSENITNNAKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRGGGSGGSGGSEQKLISEEDLGGSGGSGGSNAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIIL IMLWQKKPR**(k) BG505_MD39_gp160-dCT_CP2_GRSF4.1 (SEQ ID NO: 111) VSLGFLGAAGS TMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLK DT HWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTGGSGVTVYYGV PVWK D AET T LFCAS DAKAYETEKHN V WATHACVPTDPNSSEIHLENVTEE FNMWKN N MVEQMHEDIISLWDQSLKPC VKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAP A GFAILKCKD K KFNGTGPCQNV STVQCTH GIKPVVSTQLLLNGSLAEEE V IIRSENITN N AKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTG D IIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRGGSGSGVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDKWASLWNWFDISNWLWYIKIFIMIVGGLIGLRIVFAVLSVIHRVRQGYSPLS** (l) BG505_MD39_gp140-PDGFR_CP2_GRSF4.2(SEQ ID NO: 112) VSLGFLGAAGS T MGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLK DTH WGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTGGSGVTVYYGV PVWK D AET T LFCAS DAKAYETEKHN V WATHACVPTDPNSSEIHLENVTEE F N MWKNNMVEQMHEDIISLWDQSLKPC VKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAP A GFAILKCKD K KFNGTGPCQNV STVQCTH GIKPVVSTQLLLNGSLAEEE V IIRSENITN N AKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTG D IIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRGGSGSGVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNNQSLLALDNGSGGGSGGSGGSEQKLISEEDLGGSGGSGGSNAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQ KKPR**(m) BG505_MD39_gp160_CP2_GRSF4.1 (SEQ ID NO: 113) VSLGFLGAAGS TMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLK DT HWGIKQLQARVLAVEHYLRDQQLLGIWGCSGKLICCTGGSGVTVYYGV PVWK D AET T LFCAS DAKAYETEKHN V WATHACVPTDPNSSEIHLENVTEE FNMWKN N MVEQMHEDIISLWDQSLKPC VKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAP A GFAILKCKD K KFNGTGPCQNV STVQCTH GIKPVVSTQLLLNGSLAEEE V IIRSENITN N AKNILVQLNTSVQINCTRPNNNTVKSIRIGPGQAFYYTG D IIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFAQSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRGGSGSGVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDKWASLWNWFDISNWLWYIKIFIMIVGGLIGLRIVFAVLSVIHRVRQGYSPLSFQTHTPNPRGLDRPERIEEEDGEQDRGRSTRLVSGFLALAWDDLRSLCLFCYHRLRDFILIAARIVELLGHSSLKGLRLGWEGLKYLWNLLAYWGRELKISAINLFDTIAIAVAEWTDRVIEIGQRLCRAFLHIPRRIRQGLERALL**

15. The non-naturally occurring protein of numbered paragraph 1comprising a gp120 with a PGT121-class germline-targeting mutation,wherein the protein comprises any one of a protein comprising thesequence of any one of FIG. 9-11 or 14.

16. A protein having at least 90% homology or identity with the sequenceof the protein of any one of numbered paragraphs 2 to 15.

17. A protein having at least 95% homology or identity with the sequenceof the protein of any one of numbered paragraphs 2 to 16.

18. A monomeric protein of any one of numbered paragraphs 2 to 15.

19. The protein of any one of numbered paragraphs 2-18 furthercomprising a tag for purification or biotinylation.

20. The protein of numbered paragraph 19 wherein the tag forpurification is a his tag.

21. The protein of numbered paragraph 19 wherein the tag forbiotinylation is an avi-tag.

22. The protein of any one of numbered paragraphs 2-21 furthercomprising an additional cysteine.

23. The protein of any one of numbered paragraphs any one of numberedparagraphs 2-22 fused to a multimerization motif.

24. A nucleic acid encoding the protein of any one of numberedparagraphs 2 to 23.

25. A nucleic acid having at least 90% homology or identity with thesequence of the nucleic acid of numbered paragraph 24.

26. A nucleic acid having at least 95% homology or identity with thesequence of the nucleic acid of numbered paragraph 24.

27. The nucleic acid of any one of numbered paragraphs 23-26 wherein thenucleic acid is a mRNA.

28. A method for eliciting an immune response comprising systemicallyadministering to an animal in need thereof an effective amount of theprotein of any one of numbered paragraphs 2-23.

29. The method of numbered paragraph 28, wherein the animal is a mammal.

30. The method of numbered paragraph 29, wherein the mammal is a human.

SUPPLEMENTAL REFERENCES

-   Adams, P. D., Afonine, P. V., Bunkoczi, G., Chen, V. B., Davis, I.    W., Echols, N., Headd, J. J., Hung, L. W., Kapral, G. J.,    Grosse-Kunstleve, R. W., et al. (2010). PHENIX: a comprehensive    Python-based system for macromolecular structure solution. Acta    Crystallogr D 66, 213-221.-   Hoffenberg, S., Powell, R., Carpov, A., Wagner, D., Wilson, A.,    Kosakovsky Pond, S., Lindsay, R., Arendt, H., Destefano, J., Phogat,    S., et al. (2013). Identification of an HIV-1 clade A envelope that    exhibits broad antigenicity and neutralization sensitivity and    elicits antibodies targeting three distinct epitopes. J Virol 87,    5372-5383.-   Kulp, D. W., Subramaniam, S., Donald, J. E., Hannigan, B. T.,    Mueller, B. K., Grigoryan, G., and Senes, A. (2012). Structural    informatics, modeling, and design with an open-source Molecular    Software Library (MSL). Journal of computational chemistry 33,    1645-1661.-   McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D.,    Storoni, L. C., and Read, R. J. (2007). Phaser crystallographic    software. J Appl Crystallogr 40, 658-674.-   Murshudov, G. N., Skubak, P., Lebedev, A. A., Pannu, N. S.,    Steiner, R. A., Nicholls, R. A., Winn, M. D., Long, F., and    Vagin, A. A. (2011). REFMAC5 for the refinement of macromolecular    crystal structures. Acta Crystallogr D 67, 355-367.-   Ogura, T., Iwasaki, K., and Sato, C. (2003). Topology representing    network enables highly accurate classification of protein images    taken by cryo electron-microscope without masking. Journal of    structural biology 143, 185-200.-   Otwinowski, Z., and Minor, W. (1997). Processing of X-ray    diffraction data collected in oscillation mode. Method Enzymol 276,    307-326.-   Quan, J., and Tian, J. (2014). Circular polymerase extension    cloning. Methods in molecular biology 1116, 103-117.-   Ringe, R. P., Sanders, R. W., Yasmeen, A., Kim, H. J., Lee, J. H.,    Cupo, A., Korzun, J., Derking, R., van Montfort, T., Julien, J. P.,    et al. (2013). Cleavage strongly influences whether soluble HIV-1    envelope glycoprotein trimers adopt a native-like conformation.    Proceedings of the National Academy of Sciences of the United States    of America 110, 18256-18261.-   Salmon, P., and Trono, D. (2007). Production and titration of    lentiviral vectors. Current protocols in human genetics/editorial    board, Jonathan L Haines [et al] Chapter 12, Unit 12 10.-   Schiffner, T., de Val, N., Russell, R. A., de Taeye, S. W., de la    Pena, A. T., Ozorowski, G., Kim, H. J., Nieusma, T., Brod, F., Cupo,    A., et al. (2016). Chemical Cross-Linking Stabilizes Native-Like    HIV-1 Envelope Glycoprotein Trimer Antigens. Journal of virology 90,    813-828.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theabove paragraphs is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined in the appended claims.

What is claimed is:
 1. A non-naturally occurring protein comprising aType I immunogen, wherein the Type I immunogen is a gp120 trimercomprising an amino acid sequence selected from the group consisting of:(a) BG505-gp120-L111A-2_T135A_T139I_mC (BG505-gp120 3mut) (SEQ ID NO: 1)VWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISAWDQSLKPCVKLTPLCVTLQCTNVANNIIDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEP(b) BG505-gp120-L111A-2_5mut_mC (BG505-gp120 5mut) (SEQ ID NO: 3)VWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISAWDQSLKPCVKLTPLCVTLQCTNYTPNLTNDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEP(c) BG505-gp120-L111A-2_7mut_mC (BG505-gp120 7mut or BG505 gp120 7MUT)(SEQ ID NO: 5) VWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISAWDQSLKPCVKLTPLCVTLQCTNYAPNLINDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEP(d) BG505-gp120-L111A-2_10mut_mC(BG505-gp120 10mut or BG505 gp120 10MUT) (SEQ ID NO: 7)VWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISAWDQSLKPCVKLTPLCVTLQCTNYAPFLINDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYAFGDIIGDIRMAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEP(e) BG505-gp120-L111A-2_10mut2A_mC (SEQ ID NO: 9)VWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISAWDQSLKPCVKLTPLCVTLQCTNYAPFLINNMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYAFGDIIGDIRMAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEP and(f) BG505-gp120-L111A-2_11mut2A_mC (SEQ ID NO: 11)VWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISAWDQSLKPCVKLTPLCVTLQCTNYAPNLLSNMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYAFGDIIGDIRMAHCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSIVLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEP.


2. A monomeric protein of claim
 1. 3. The protein of claim 1, furthercomprising a tag for purification or biotinylation.
 4. The protein ofclaim 3, wherein the tag for purification is a his tag.
 5. The proteinof claim 3, wherein the tag for biotinylation is an avi-tag.
 6. Theprotein of claim 1, further comprising an additional cysteine.
 7. Theprotein of claim 1 fused to a multimerization motif.